EPA/600/R-16/061 I May 2016
www.epa.gov/homeland-security-research
United States
Environmental Protection
Agency
oEPA
Attenuation of Ricin Toxin under Ambient
Conditions and Elevated Temperature and
Humidity
Office of Research and Development
National Homeland Security Research Center
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EPA/600/R-16/061
May 2016
REPORT
Attenuation of Ricin Toxin
under Ambient Conditions and
Elevated Temperature and
Humidity
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
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Disclaimer
The U.S. Environmental Protection Agency (EPA), through its Office of Research and
Development's (ORD's) National Homeland Security Research Center (NHSRC), funded,
directed and managed this work through Contract Number EP-C-11-038, Task Order 0017, with
Battelle. This report has been peer and administratively reviewed and has been approved for
publication as an EPA document. The views expressed in this report are those of the authors and
do not necessarily reflect the views or policies of the Agency. Mention of trade names or
commercial products does not constitute endorsement or recommendation for use of a specific
product.
Questions concerning this document or its application should be addressed to:
Mr. Joseph Wood
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Mail Code E343-06
Research Triangle Park, NC 27711
919-541-5029
in
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Acknowledgments
Contributions of the following individuals and organization to this report are gratefully
acknowledged:
U.S. Environmental Protection Agency (EPA) Project Team
Joseph Wood, Principal Investigator; National Homeland Security Research Center
Richard Rupert, On-Scene Coordinator, Region 3
Lawrence Kaelin, Office of Emergency Management
US EPA Technical Reviewers of Report
Shannon Serre, Office of Emergency Management
Charlie Fitzsimmons, On-Scene Coordinator, Region 3
Alden Adrion, Oak Ridge Institute for Science and Education Post-Doctoral Fellow
US EPA Quality Assurance
Eletha Brady Roberts, National Homeland Security Research Center
Battelle
iv
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Executive Summary
The U.S. Environmental Protection Agency (EPA), Office of Research and Development (ORD),
Homeland Security Research Program (HSRP) is striving to protect human health and the
environment from adverse impacts resulting from acts of terror by investigating the effectiveness
and applicability of technologies for homeland security-related applications. This report presents
the results of an investigation to evaluate the attenuation of ricin toxicity on indoor materials as a
function of environmental conditions.
This study focused on the attenuation of ricin toxin on six types of materials representative of a
mail sorting facility and/or indoor building materials. Attenuation tests were conducted under
various combinations of temperature, relative humidity (RH), and contact time, using two forms
of ricin toxin: a commercially-available "pure" preparation and a "crude" form of the toxic
material prepared in the laboratory from castor beans.
Summary of Major Findings
Overall, the crude ricin was more persistent than the pure form of the toxin. Average two-week
attenuation of the crude ricin at temperatures between 20-30 °C (temperatures expected to be
achievable with the HVAC system of the building) ranged from 7% (at 20 °C/45% RH) to 81%
(at 30 °C/45% RH). After 28 days at 20 °C/45% RH, the crude ricin averaged 77% attenuation
across the six materials tested.
For the pure ricin, heat treatments at the elevated temperatures of 40 °C for 5 days and 50 °C for
2-3 days achieved greater than 96% attenuation on mild steel. For the crude ricin preparation,
appreciable recovery of the ricin still occurred at 40 °C after two weeks. A seven-day heat
treatment at 50 °C was required to achieve greater than 98% attenuation of the crude ricin on
mild steel.
From the statistical analyses, increasing temperature either had no significant effect on
attenuation, or there was an increase in attenuation associated with increasing temperature. The
effect of increasing temperature on attenuation was more pronounced when comparing results
with a 10 °C temperature difference. Under the environmental condition most resembling the
indoor environment (20 °C, 45% RH), there was no significant attenuation of the crude ricin at
14 days on any of the materials except mild steel. At 30 °C, the average ricin attenuation across
all materials at 14 days ranged from 39-81%.
Table ES-1 provides another perspective of the study results in terms of highlighting the fact that
there were only seven cases (out of over 200 test combinations of ricin type, temperature, RH,
material, and contact time) in which we observed greater than 99% attenuation of ricin. As seen
in this table, in general, elevated temperature or RH was required to achieve greater than 99%
reduction of pure or crude ricin toxin. More specifically, there were no cases in which any form
of ricin was attenuated more than 99% at 20 °C or at 25 °C/45% RH. There was only one case
where the crude ricin preparation was attenuated more than 99%. Attenuation of more than 99%
occurred most often on the mild steel and paper materials.
While in many of the tests there was no significant effect of material on the attenuation of ricin, in the
cases where there was a significant effect, ricin was generally attenuated most on mild steel and the
least on wood.
v
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In general, increasing the RH level from 45 to 75% did not significantly affect attenuation of ricin.
The effect of RH (or lack thereof) was similar for both ricin types, while the effect of RH appeared to
be somewhat dependent on the material. For example, for both the mild steel and wood materials,
there was either no effect of RH, or an increase in RH significantly increased the ricin attenuation in
nearly all of the comparisons. The opposite effect of RH occurred with the plastic and rubber
materials.
The results showed that the crude ricin was more stable, i.e., more difficult to attenuate, than the
pure ricin in most of the tests conducted in this study. The attenuation results for the crude ricin
also exhibited more variability than the attenuation results for the pure ricin. The higher
variability of results and stability of the crude ricin may be due to the presence of additional
proteins and other organic materials in the crude suspension. These potentially extraneous
proteins and other materials (e.g., carbohydrates, fatty acids, ash) could have mitigated the
effects of the environmental conditions, as well as interfered in the quantitation assay, and would
be absent in the commercially available pure material. The use of a biological system (a cell-
based assay) to quantitate ricin toxicity, regardless of ricin type, may also have contributed to
variability in results.
Table ES-1. Test Parameter Combinations Demonstrating Over 99% Attenuation of Ricin
Test
Ricin
Form/Target
Mass
Temp °C
%RH
Contact
Time Material
(Days)
% Reduction
± 95%
Confidence
Interval
4
Pure/250
25 °C
75%
7
Mild Steel
99.87 ±0.11
Crude/320 |ig
7
Paper
99.54 ±0.12
5
Pure/250 |ig
25 °C
75%
14
Mild Steel
99.95 ±0.03
6
Pure/250 |ig
30 °C
45%
7
Pine Wood
99.38 ±0.47
7
Paper
99.83 ±0.24
\T
Pure/250 |ig
50 °C
20%
6
Mild Steel
99.05 ± 0.48
7
Mild Steel
99.92 ±0.02
Detailed data from each test number can be referenced in Appendix A.
vi
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Contents
Acknowledgments iv
Executive Summary v
1.0 Introduction 1
2.0 Procedures 3
2.1 Test Matrix 3
2.2 Ricin Toxin 4
2.3 Test Materials 4
2.4 Inoculation of Coupons 5
2.5 Environmental Test Chamber and Procedures 6
2.6 Coupon Extraction and Ricin Toxin Quantification 8
2.7 Attenuati on C al cul ati on 11
2.8 Statisitical Analysis 12
2.9 Surface Damage 13
3.0 Quality Assurance/Quality Control 15
3.1 Equipment Calibration 15
3.2 QC Results 15
3.3 Audits 15
3.4 QA/QC Reporting 16
3.5 Data Review 16
4.0 Summary of Results and Discussion 17
4.1 Test Environmental Conditions 17
4.2 Recovery of Ricin from Positive Controls 18
4.3 Environmental Conditions Required for 99% Attenuation 18
4.4 Attenuation Results for Tests at 20-30 °C 19
4.5 Attenuation Results for Tests at 40 and 50 °C 24
4.6 Statistical Analyses to Assess Effect of Test Variables on Attenuation 27
4.7 Summary 29
5.0 References 31
vii
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Figures
Figure 2-1. Coupon Types from Left to Right: Mild Steel, Neoprene Rubber, Optical Plastic,
Pine Wood, Industrial Carpet, Paper 5
Figure 2-2. Liquid Inoculation of Coupon Using a Micropipette 6
Figure 2-3. Schematic Diagram of Environmental Exposure Chamber 7
Figure 2-4. Representative Graph of Temperature and RH Stability (Test 10) 8
Figure 2-5. Visual Demonstration of MTT Assay on a Microplate 10
Figure 2-6. Example of Ricin Cytotoxic Profile with Corresponding Absorbance Measured
Using a Microplate Reader 10
Figure 4-1. Summary of Average Percent Recovery from Positive Controls for Pure Ricin and
Crude Ricin by Material Type ± Standard Deviation 18
Figure 4-2. Summary of Percent Reduction (Tests 1-14) Results, for Steel and Rubber 21
Figure 4-3. Summary of Percent Reduction (Tests 1-14) Results, for Plastic and Wood 22
Figure 4-4. Summary of Percent Reduction (Tests 1-14) Results, for Carpet and Paper 23
Figure 4-5. Summary of Percent Reduction (Tests 15) Results at 50 °C, by Time, Comparing
Pure and Crude Ricin ± 95% Confidence Interval 25
Figure 4-6. Summary of Percent Reduction (Tests 16) Results at 40 °C, by Day, Comparing
Pure and Crude Ricin ± 95% Confidence Interval 25
Figure 4-7. Summary of Percent Reduction (Tests 17) Results, by Time, Comparing Pure and
Crude Ricin ± 95% Confidence Interval 26
Figure 4-8. Summary of Percent Reduction (Tests 18) Results at 40 °C, by Day, Comparing
Pure and Crude Ricin ± 95% Confidence Interval 26
Figure 4-9. Comparison of Average Percent Reduction for Pure Ricin and Crude Ricin by
Material Type ± Standard Deviation, for Tests 1-14 29
Tables
Table ES-1. Test Parameter Combinations Demonstrating over >99% Attenuation of Ricin vi
Table 2-1. Attenuation Test Matrix 3
Table 2-2. Test Materials 4
Table 2-3. Average Dilution Factors per Coupon Material 11
Table 3-1. Performance Evaluation Audits 15
Table 4-1. Actual Attenuation Conditions for Environmental Chamber Tests 17
Table 4-2. Test Parameter Combinations Demonstrating Greater than 99% Reduction of Ricin
Materials 19
Table A-l. Attenuation of Pure Ricin Toxin A-l
Table A-l. Attenuation of Pure Ricin Toxin (Continued) A-2
Table A-l. Attenuation of Pure Ricin Toxin (Continued) A-3
Table A-2. Attenuation of Crude Ricin Toxin A-4
Table A-2. Attenuation of Crude Ricin Toxin (Continued) A-5
Table A-2. Attenuation of Crude Ricin Toxin (Continued) A-6
viii
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List of Appendices
Appendix A. Detailed Test Results
Appendix B. Detailed Statistical Analysis
ix
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Abb re vi ati on s/ Acronym s
4-PL four-parameter logistic
ANOVA Analysis of variance
ASTM American Society of Testing and Materials
BSC biological safety cabinet
CI confidence interval
cm centimeter(s)
°C degree(s) Celsius
E-beam electron beam
EPA U.S. Environmental Protection Agency
h hour
HSRP Homeland Security Research Program
HVAC Heating, ventilation, and air conditioning
IV intravenous
kg kilogram(s)
kGy kilogray(s)
L liter(s)
LD50 median lethal dose; individual dose required to kill
50 percent of a population of test animals
LOD limit of detection
l_ig microgram(s)
l_iL microliter(s)
mg milligram(s)
mL milliliter(s)
mil thousandth of an inch
min minute(s)
MTT 3-(4,5-dimethlythiazol-2-yl)-2,5-
diphenyltetrazolium bromide
NA not applicable
ng nanogram(s)
nm nanometer(s)
NHSRC National Homeland Security Research Center
ORD Office of Research and Development
PBS phosphate buffered saline
QA quality assurance
QAPP Quality Assurance Project Plan
QC quality control
QMP Quality Management Plan
RH relative humidity
rpm revolution(s) per minute
SD standard deviation
x
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SE standard error
SFW sterile filtered water (cell-culture grade)
STREAMS Scientific, Technology, Research, Engineering, and
Modeling Support
TO time zero
TSA technical systems audit
USAMRIID United States Army Medical Research Institute of
Infectious Diseases
xi
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1.0 Introduction
The U.S. Environmental Protection Agency Office of Research and Development's (ORD's)
Homeland Security Research Program (HSRP) is helping protect human health and the
environment from adverse impacts resulting from the release of chemical, biological, or
radiological agents. With an emphasis on decontamination and consequence management, water
infrastructure protection, and threat and consequence assessment, the HSRP is working to
develop technology and information that will help detect the intentional introduction of chemical
or biological contaminants in buildings or water systems; contain these contaminants;
decontaminate buildings, water systems, or other infrastructure; and facilitate the disposal of
material resulting from restoration activities.
In 2013, several letters that contained ricin toxin were sent to various locations, including the
White House and the office of the New York City mayor (according to the U.S. Attorney's
Office in a memorandum dated June 28, 2013). These contaminated letters had the potential to
contaminate the corresponding mail-sorting facilities and equipment, creating an exposure risk
for those working in the area. Ricin toxin is a highly toxic protein produced within the beans of
the Ricinus communis (castor bean) plant. The median lethal dose (LD50) in mice is 5
micrograms per kilogram (|ig/kg) via intravenous (IV) injection.(1) Extrapolations have been
made that indicate a human LD50 exposure could be ~1 to 5 milligrams per kg (mg/kg) IV. The
ricin aerosol LD50 for nonhuman primates is estimated to be 10-15 (ig/kg.
In this investigation, the attenuation of ricin toxin activity (pure and crude preparations) over
time was evaluated under varying environmental conditions to help determine conditions under
which further decontamination may be needed. The majority of the tests were conducted with
multiple materials under temperature conditions relatively easily obtainable (20-30 degrees
Celsius [°C]) with a building's heating, ventilation, and air conditioning (HVAC) system.
Relative humidity (RH) levels in these tests were controlled to either 40 or 75%.
In the last four experiments of the study, we assessed the attenuation of ricin at elevated
temperatures (with no RH control) where additional heating equipment would probably be
needed (40 and 50 °C), but the elevated temperatures would not be expected to be overly
detrimental to the interior materials. In these last four tests, we used just one material (mild
steel), which allowed us to assess multiple time points in one experiment.
The present study discussed in this report builds on an earlier EPA HSRP study(2) in which the
attenuation of pure ricin was assessed for just a few materials and a few environmental
conditions. We were unable to identify any other scientific literature describing the attenuation of
ricin on material surfaces for the more benign air temperatures we studied. While the U.S. Army
Medical Research Institute of Infectious Diseases (USAMRIID) reports that ricin is stable for
only an hour at 50 °C at a pH of 7.8(3), no data or references were provided for this assertion.
Also, since pH was reported, it is assumed the USAMRIID information is applicable only to
ricin stability in aqueous liquids. Other researchers have similarly reported on the persistence of
ricin in foods or liquids at elevated temperature treatments achieved through means such as
boiling or autoclaving.(4'5) Lastly, another source mentioned that pure and crude ricin on
materials could be effectively denatured at 82-88 °C, 80-85% RH, for 24 hours.(6) None of the
aforementioned research investigated the attenuation of ricin on solid surfaces at air temperatures
that could be used in a building.
1
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The results of this investigation attempt to fill some of the data gaps discussed above, by
providing stakeholders with high quality, peer-reviewed data on how ricin may attenuate over
time as a function of the type of ricin preparation, the material the ricin is associated with,
temperature, and RH. The materials used in this study include those associated with mail
sorting equipment such as mild steel, neoprene rubber, paper, and optical grade plastic, while
bare pine wood and industrial carpet were used to represent building materials.
2
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2.0 Procedures
This section provides an overview of the procedures used for the evaluation of controlled and
ambient environmental parameters to naturally attenuate both pure and crude forms of ricin toxin
on six different materials. Testing was performed in accordance with the peer-reviewed and
EPA-approved Quality Assurance Project Plan (QAPP) for the Neutralization of Ricin Toxicity
Using Elevated Temperature and Humidity,(7) The QAPP provides additional procedural details
that are not included in this report. Procedures were also consistent with a previous study
examining the efficacy of hydrogen peroxide vapor to neutralize ricin.(8)
2.1 Test Matrix
The test matrix for the attenuation tests is shown in Table 2-1. Tests 1 -14 used the full set of
materials included in this study while Tests 15 through 18 were evaluated utilizing a
downselected material (mild steel). We chose to use mild steel in the last four tests since this
hard nonporous material exhibited less variability in ricin recoveries among its replicates then
the other materials. The use of only one material in these last four trials also allowed us to assess
ricin recovery at multiple timepoints, with all test coupons using the same positive controls.
Table 2-1. Attenuation Test Matrix
Tom
Number
Malerials
laruel Kielii
Mass
IjlU)
l a rue I
Temp
(
1 a rueI
"..KM
Time (l)a\si
1
30
75
7
2
25
45
7
3
14
4
25
75
7
5
Mild Steel
14
6
Rubber
30
45
7
7
Plastic
14
8
Wood
Pure 250
Crude 250
30
75
14
9
Carpet
20
45
7
10
Paper
14
11
20
75
7
12
14
13
20
45
21
14
28
15
50
6,24,30,48,72, and 96 hours (h)
16
Mild Steel*
40
20"
48,72,96,120,144, and 168 h
17
50
48,72,96,120,144, and 168 h
18
Crude 250
40
3,4,5,6,7,10,11,12,13,14 days
Only one material tested to allow for multiple time points per test.
**RH monitored but not controlled, average value shown.
3
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2.2 Ricin Toxin
Testing was conducted with a commercially-available preparation of pure ricin toxin (Cat. No.
L-1090: Ricin communis agglutinin II, 5 mg per milliliter [mg/mL] protein concentration, Vector
Laboratories, Burlingame, CA), which was stored at 2 to 8 °C and used as received. In addition,
a crude preparation of the toxin was extracted from whole castor beans obtained from Vector
Laboratories (Vector Laboratories, Inc.). The crude preparation of ricin toxin was performed in
the laboratory using methods derived from the scientific literature,(9) and this batch was used
throughout the study. Briefly, whole castor beans were de-husked and homogenized into a slurry,
precipitated from the solution, dialyzed, and rinsed with sterile phosphate buffered saline (PBS
[Cat #D8537 Sigma-Aldrich, St. Louis, MO]). The final crude ricin toxin was prepared in sterile
PBS and stored at 2 to 8 °C. While our target ricin titer for the crude preparation was 5 mg/mL,
the actual titer averaged 6.4 mg/mL based on post-test statistical analyses.
2.3 Test Materials
The test materials included mild steel, neoprene rubber, optical plastic, pine wood, industrial
carpet, and paper. Information on these materials is presented in Table 2-2, and a picture of each
is presented in Figure 2-1. Material coupons were cut to uniform length and width (Table 2-2)
from larger pieces of stock material. Materials were prepared for testing by either sterilization
via electron beam (E-beam) irradiation at -200 kilograys (kGy; E-beam Services Inc., Lebanon,
OH) or autoclaved at 121 °C for 15 minutes (min). E-beam-irradiated material coupons were
sealed in 6 mil (0.006 inch) Uline Poly Tubing (Cat. No. S-2940, Uline, Chicago, IL), and
autoclaved coupons were sealed in sterilization pouches (Cat. No. 01-812-50, Fisher, Pittsburgh,
PA) to preserve sterility until the coupons were ready for use. Sterilization was intended to
eliminate contamination by microorganisms that might interfere with the cell-based assay used to
assess ricin bioactivity.
Table 2-2. Test Materials
Miiloriiil
l.ol. liiilch. AS'l'M No., or
Ohsm iilion
MiinuliiclumV
Supplier Name
Locution
Approximate Coupon
Si/o. \\ id 111 \ l.iiiiilh \
Thickness
Miilcriiil
I'lvpiii'iilion
Mild
(Carbon)
Steel
Gauge 12
Adept Products,
West Jefferson, OH
1.9 centimeters (cm) x 7.5
cm x 0.2 cm
Autoclave
Neoprene
Rubber
Nonmarking Neoprene Rubber
Part # 8837k214
McMaster Can-
Aurora, OH
1.9 cm x 7.5 cm x 0.3 cm
E-Beam
Optical
Grade
Plastic
Optically Clear Cast Aciylic Sheet
McMaster Item #8560K263
McMaster Can-
Aurora, OH
1.9 cm x 7.5 cm x 0.3 cm
E-Beam
Pine Wood
Item #: 3542 Model #: 142 8PINE
Lowes
Hilliard, OH
1.9 cm x 7.5 cm x 0.3 cm
E-Beam
Carpet
Shaw Swizzle EcoWorx, Style: 10401
Color: Jacks
Shaw Industries
Dalton, GA
1.9 cm x 7.5 cm x 0.7 cm
E-Beam
Paper
Boise Aspen Laser Paper 24 pounds
Part #BPL-2411 -RC
Office MaxC
Hilliard, OH
1.9 cm x 7.5 cm x 0.3 cm
E-Beam
4
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Figure 2-1. Coupon Types from Left to Right: Mild Steel, Neoprene Rubber, Optical
Plastic, Pine Wood, Industrial Carpet, Paper
2.4 Inoculation of Coupons
Test and positive control coupons were placed on a flat surface within a Class II biological safety
cabinet (BSC) and inoculated individually with a target mass of approximately 250 jig of either
the purified or crude ricin toxin. The mass quantity of pure ricin toxin inoculated was 250 jig per
material based on certified titer of stock material as received from the vendor. Actual delivered
mass of crude ricin toxin per coupon material was determined using a cell-based bioassay (see
Section 2.6) and averaged approximately 320 jig per coupon for the study. While this average
actual quantity of crude ricin applied to each coupon was greater than our target of 250 jig, ricin
attenuation was calculated based on the actual recovery of mass from positive controls from each
test. The higher than expected crude ricin inoculum levels may be attributed to the variability of
the cell-based assay at the higher dilutions, the variability of ricin content associated with using
actual castor beans, and potential bias from additional proteins in the crude suspension.
A 50 microliter (jiL) inoculum of either the purified (5 mg/mL) or crude ricin (6.4 mg/mL) toxin
stock suspension was dispensed using a micropipette and applied as a single streak across the
coupon surface (Figure 2-2). This technique provided decreased drying times and enabled greater
distribution of toxin across the coupon surface as compared to a single drop of the suspension.
After inoculation, the coupons were transferred to a Class III BSC and left undisturbed to dry for
approximately one hour (h) (or until visually dry) under ambient conditions, -22 °C and 40%
RII
5
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Figure 2-2. Liquid Inoculation of Coupon Using a Micropipette
The number and type of replicate coupons used for each combination of material and
environmental condition included were:
• Five test coupons (inoculated with ricin toxin and exposed to experimental
temperature/RH for the test duration)
• Five positive controls (inoculated with ricin toxin and extracted after 1 h drying time)
• One laboratory blank (not inoculated and not exposed to experimental temperature/RH)
• One procedural blank (not inoculated and exposed to experimental temperature/RH).
Approximately 1 h post-inoculation (or until materials were visibly dry), coupons intended for
attenuation testing (including blanks) were transferred into the test chamber and exposed to the
environmental conditions using the environmental test chamber and application conditions
specified in Section 2.5. Positive controls were then extracted and analyzed.
2.5 Environmental Test Chamber and Procedures
Figure 2-3 shows a schematic diagram of the bench-scale exposure chamber. Attenuation testing
was conducted inside the approximately 38 liter (L) stainless steel chamber. The chamber was
insulated to prevent condensation on the inside walls. As a means of secondary containment and
considering laboratory personnel safety, this test chamber was housed inside a custom acrylic
compact glove box (Plas Labs, Inc., Lansing, MI) that was hard-ducted to the facility exhaust
system to maintain negative pressure.
6
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Plas-Labs Glovebox
Vaisala
Water Bath
Water Bath
Radiator
— Indicates water lines
Indicates Air Lines
Indicates electrical lines to and from RH, temperature
Figure 2-3. Schematic Diagram of Environmental Exposure Chamber
Temperature was controlled using an external water bath connected to a heat exchanger within
the test chamber. RH was controlled using an external water bath connected to a Nafion tube
pervaporation system (Perma Pure; Lakewood, NJ) and through the use of fixed humidity point
salts (Sigma-Aldrich; St. Louis, MO).( 0) Temperature and RH in the test chamber were
measured using an HMT368 temperature and humidity probe (Vaisala, Inc., Woburn, MA) and
controlled with a CN1-822 controller (Omega Engineering, Stamford, CT). Data were recorded
every minute during the experimental temperature/RH exposure time using the controller-
associated Omega Engineering iLOG software. A typical representative graph of the
environmental conditions (Test 10) data collection can be seen in Figure 2-4.
7
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Test #10 Persistence
100,00
70.00
60.00
w
"E
z>
% 50.00
30.00
20.00
10.00
0.00
H-
^ J? J? a* J? j? J* j? ^ J? j? J? j? j* J? jt j?
<$ <$ <$ <5^ & <5^ N <$>v <& <& <$>N
Time
I 1 1 1 I
H-
-t-
H-
H-
I 1 1 ' I 1 1 1 I 1 1 1 I
I ' 1 1 I
H
-Temp, °C
-RH, %
Figure 2-4. Representative Graph of Temperature and RH Stability (Test 10; shaded
portion of graph shows time period for test)
2.6 Coupon Extraction and Ricin Toxin Quantification
At time zero (TO) (positive controls and blanks) and each non-zero time point, the coupons and
blanks were individually placed in 50 mL conical tubes containing 10 mL of sterile PBS for
extraction. The vials were capped, placed on their sides and agitated on an orbital shaker for 15
min at approximately 200 revolutions per minute (rpm) at room temperature. The presence of
residual active toxin in the test and control coupon extracts was determined using the bioassay
described below.
The mechanism of action by which ricin toxin exerts its toxic effect is through inhibition of
protein synthesis within cells. Such inhibition of protein production leads to cell death.
Therefore, an in vitro cytotoxicity assay was used to evaluate the level of bioactive ricin toxin
extracted from both attenuated and positive control material coupons. The bioassay used in this
evaluation for determining the cytotoxicity (concentration) of bioactive ricin toxin is based on
the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay developed by
Mosmann.' "' Cytotoxicity is reported as mass of bioactive toxin as determined using a reference
standard prepared from the purified form of ricin toxin.
8
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To conduct this MTT assay, Vero cells (ATCC; Manassas, VA; kidney epithelial cells from the
African green monkey) were seeded in wells of a 96-well microplate at a density of
approximately 2 x io4 cells/well. Cells were then incubated for approximately 18 to 30 h at 37 ±
2 °C under 95% air and 5% carbon dioxide and exposed to the various coupon extracts (test,
positive controls and blank controls) by adding 100 |iL extract or test dilution to each well and
performing a series of two-fold dilutions down each plate. Following 48 to 72 h exposure to
sample extracts, the cells were incubated in the presence of MTT, where mitochondrial enzymes
convert the yellow MTT to a purple formazan salt. The absorbance of this purple reaction
product, read at 570 nanometers (nm) using a SPECTRAmax PLUS 384 microplate reader
(Molecular Devices, Sunnyvale, CA), is directly proportional to the number of living cells and
inversely proportional to the cytotoxic potential of ricin toxin (Figures 2-5 and 2-6). For all
dilutions and sample transfers into the individual wells of a 96-well plate (Fisher Scientific;
Pittsburgh, PA), a micropipette (Mettler-Toledo Rainin; Oakland, CA) was used with the pipette
tip was placed between wells to ensure that cross contamination did not occur.
To determine the concentration of ricin toxin from each test sample, a pure ricin toxin standard
(Vector Laboratories, Inc.) was prepared from the commercially-available stock solution and
assayed in parallel on each test plate. The pure ricin toxin stock solution was used to prepare a
seven point-standard curve of absorbance versus calculated mass of ricin toxin protein. For each
standard and test sample, absorbance values of the reference wavelength (630 nm) were
subtracted from the absorbance values at 570 nm for each well. For each point used in generating
the standard curve, the mean absorbance values (Y-axis) were plotted against the concentration
in nanograms (ng)/mL, and a four-parameter logistic (4-PL) curve was generated by the SoftMax
Pro Version 4.7 software included in the SPECTRAmax PLUS 384 microplate reader using the
equation:
(max- mm)
Y = 1111IH r-
1+(X/C)J
where:
Y = absorbance %:
X = concentration of ricin iig-mL;
max = Y-value of the asymptote at the low values of X absorb,nice:
mill = Y-value of the asymptote ai the high values of X °o absorbance:
B = value related] to the slope of the curve between the asymptotes;
C = X-value of die midpoint between max and mm ng/mL
(1)
9
-------
Purple = cells alive;
little to no toxin
Increasing ricin
concentration
Yellow = cells dead;
abundant toxin
Figure 2-5. Visual Demonstration of MTT Assay on a Microplate
A 5 -—-
=
03
O
=
ra
a
~
V)
a
"4
I
£
Ricin Standard Curve
pie = alive? cells; littl
to ilo toxin
I I I I
0.1
Yellow = dead tells;
abundant floxin
, ! ! 1 1 I ,
I I I I I
1
Protein (ng/mL)
10
Figure 2-6. Example of Ricin Cytotoxic Profile with Corresponding Absorbance
Measured Using a Microplate Reader
10
-------
Throughout the study, the inherent cytotoxicity of material coupon extracts from laboratory and
procedural blank coupons was assessed to determine a starting dilution that could mitigate any
potential confounding cytotoxic effects observed in the ricin bioassay. To account for this
potential for coupon extract-induced cytotoxicity in the ricin bioassay, the dilution factor of
coupon extracts exhibiting cytotoxicity of less than 20%, when compared to negative controls
(cell culture medium only ), were selected as the starting dilution for all test samples. The average
dilution schemes that effectively baselined the cytotoxicity of the test coupons are shown in
Table 2-3.
Table 2-3. Average Dilution Factors per Coupon Material
Material
Dilution Factors Required to "Zero Out" Coupon
Cytotoxicity
Mild Steel
1:10
Neoprene Rubber
1:25
Optical Grade Plastic
1:8
Pine Wood
1:7
Industrial Carpet
1:135
Paper
1:8
2.7 Attenuation Calculation
The attenuation of ricin was assessed by determining the mass of bioactive toxin extracted from
each test coupon subjected to specified environmental conditions as compared to the average
mass of bioactive toxin extracted from the 1 h (TO) positive control coupons.
Attenuation in terms of percent reduction for a given environmental condition, material, and ricin
type, was calculated as the difference between the mean control mass values and the mean test
mass values, divided by the mean control mass values, i.e.:
(2)
Masse-; — Masst-, 1 Masst.. ,
- 100 % = ( I- - * 100 %„
MassCy X Massci}!
where Masse,/ refers to the j individual mass values obtained from the positive control coupons,
Masst.,; refers to the j individual mass values obtained from the corresponding test coupons, and
the overbar designates a mean value. In this study, there were five positive controls and five
corresponding test coupons (i.e.J = 5) for each coupon material.
In samples where no bioactive toxin was observed in any of the five test coupon extracts after
attenuation, an adjusted limit of detection (LOD) value for that material was assigned. The
adjusted LOD was defined as mass of ricin toxin that corresponded to the lowest dilution factor
in the standard curve.
11
-------
s rt
\Masst:"
(
. a. )
i—— 2
} Masse-
| 2
\Mll8Stj
MassCi" /
The variance of the mean percent reduction was estimated through propagation of error using
Taylor series approximation. Let 2 be the variance of the five positive control coupons, and
let 2 be the variance of the five test coupons. Then the estimated standard error (SE) of
percent reduction is:
\Masst~ ( S~t(
(3)
100%.
V 3
where the number 5 represents the number j of coupons in both the control and test data sets.
Each attenuation result is reported as a mass value with an associated 95% CI, calculated as
follows:
95 % CI = Attenuation (% Mass Reduction) ± (1.96 x SE) (4)
Significant differences in attenuation for the different test conditions and toxin types may be
assessed visually in some of the figures presented in Section 4, based on whether or not the 95%
CI values for each attenuation result overlapped. However, significant effects of test variables
were more robustly analyzed using the statistical procedures described below.
2.8 Statistical Analysis
The assumption of normality for the data set was more reasonable for the log-transformed
attenuation (percent reduction) values than the untransformed values. This assessment was
based on com pari son of the hi stogram and normal probability plots that were created for the
residuals from the ANOVA models, for both the log-transformed and untransformed values.
Thus, all models were fitted to the log-transformed values. In addition, since the primary interest
was in percent reduction from time zero, the ratio of values at each time point was taken relative
to the baseline (Day 0) mean prior to analysis. The log-transformed ratio was analyzed, but
results were transformed so that interpretation could be in terms of the percent reduction. A
smaller ratio is associated with a larger percent reduction, and a ratio greater than one is
associated with a negative percent reduction. All statistical analysis was performed using SAS
(Version 9.4; Cary, NC, USA).
Analysis of variance (ANOVA) models were fitted to the log-transformed ratios for each
combination of temperature and humidity. The models included main effects for time, material,
and ricin preparation (pure or crude). The models also included all pairwise interactions and the
three-way interaction. The following three-way ANOVA model was fitted to the base-10
log-transformed ratio response, separately for each combination of temperature and humidity:
Y ijkn = j.i + time., + material; + type* + (time*material),/ + (time*typek +
(material *type)yi; + (time* m ateri al * ty pe )?;t +%£„
where YiJkn is the observed log-transformed, baseline-adjusted value of the nth replicate for time.,,
material;, and type*. The parameter j.i is an overall constant, time., is the effect of time i, material;
is the effect of material j, type* is the effect of ricin type A\ (time*material),/ is the interaction
effect between time i and material j, (time*typek is the interaction effect between time i and
12
-------
ricin type k, (material*type)(/: is the interaction effect between material j and ricin type k,
(tirne*rnaterial*type)f//,. is the interaction effect between time material /, and ricin type k, and
8ykn is the random error unexplained by the model. Baseline (Day 0) results were not included in
the model. The three-way interaction was significant in all of the models, and thus the effect of
each factor (time, material and preparation) had to be interpreted separately at each combination
of the other two factors. The models were used to estimate the percent reduction with 95 percent
confidence for each combination of time, material and ricin type. In addition, pairwise
comparisons were performed to test for significant differences between each combination of
time, material and ricin type that differed in only one parameter. For these comparisons,
unadjusted and Tukey-adjusted p-values were reported. P-values below 0.05 indicate a
significant effect.
ANOVA models were also fitted separately for each combination of ricin type and material. The
models included main effects for temperature, humidity, and time. The models also included all
pairwise interactions and the three-way interaction. The following three-way ANOVA model
was fitted to the base-10 log-transformed ratio response, separately for each combination of ricin
type and material:
Y ijkn = [i + time, + temperature, + humidity^ + (time*temperature),7 + (time* humidity),/; +
(temperature* humidity)//,. + (time*temperature*humidity)^ +8 ykn
where is the observed log-transformed baseline-adjusted value for the nth replicate for time,,
temperature,, and humidity^. The parameter |j, is an overall constant, time, is the effect of time /,
temperature, is the effect of temperature /, humidityis the effect of humidity &,
(time*temperature),7 is the interaction effect between time i and temperature j, (time*humidity),/
is the interaction effect between time i and humidity k, (temperature*humidity)(/;; is the
interaction effect between temperature j and humidity &, (time*temperature*humidity),(/, is the
interaction effect between time /, temperature /, and humidity k, and is the random error
unexplained by the model. Only results at study day 7 and study day 14 were included in the
model with the exception of steel that included all available time points. The three-way
interaction was significant in most of the models, and thus the effect of temperature and humidity
had to be interpreted separately at each combination of the other two factors. The models were
used to perform pairwise comparisons to test for significant differences between each
combination of time, temperature and humidity that differed in only one parameter. For these
comparisons, unadjusted and Tukey-adjusted p-values were reported. For the purposes of this
report, the effects of test variables were reported as significant if the Tukey-adjusted P-values
were less than or equal to 0.05. The detailed results of these statistical analyses are presented in
Appendix B.
2.9 Surface Damage
The physical effect of environmental parameters on the materials was qualitatively monitored
during the evaluation. This approach provided a gross visual assessment of whether the
environmental state changed the appearance of the test materials. The procedural blank (coupon
that is exposed to environmental conditions, but has no toxin applied) was visually compared to a
laboratory blank coupon (a coupon not exposed to the environmental conditions and having no
13
-------
toxin applied). No obvious visible damage was observed even at temperatures of 40 and 50°C
(mild steel was only material tested at 40 and 50 °C tests), which might include structural
damage, surface degradation, discoloration, or other aesthetic impacts.
14
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3.0 Quality Assurance/Quality Control
Quality assurance (QA)/quality control (QC) procedures were performed in accordance with the
Scientific, Technology, Research, Engineering, and Modeling Support (STREAMS II) Program
Quality Management Plan (QMP), Version 3 and the QAPP.(7) The QA/QC procedures and
results are summarized below.
3.1 Equipment Calibration
All equipment (e.g., pipettes, incubators, microplate reader, biological safety cabinets) and
monitoring devices (e.g., thermometer, hygrometer) used at the time of the evaluation were
verified as being certified, calibrated, or validated.
3.2 QC Results
QC efforts conducted during testing included positive control samples (inoculated, dried for
-one h, then recovered), procedural blanks (not inoculated, attenuation), laboratory blanks (not
inoculated, no attenuation), and inoculation control samples (analysis of the stock toxin
suspension).
Positive control samples were run at the beginning of each test to determine the loss of
cytotoxicity over the -one h drying period. The amount of ricin recovered from these positive
controls was sufficient to determine percent reduction due to the cytotoxicity assay standard
range of 0.1 to 10 ng.
All procedural and laboratory blanks met the acceptance criteria by the use of dilution to mitigate
inherent material specific cytotoxicity, as previously discussed. Inoculation control samples were
taken from the purified and crude stock toxin suspension each day of testing and assayed against
the 4-PL standard curve. Using a Grubbs outlier test, the control samples were assessed, and no
outliers were found.
3.3 Audits
3.3.1 Performance Evaluation A udit
Performance evaluation audits were conducted to assess the quality of the results obtained during
these experiments. Table 3-1 summarizes the performance evaluation audits that were
performed.
Table 3-1. Performance Evaluation Audits
Measurement
Audit
Procedure
Allow sihle
loleniiiee
Aclu;il
Toler;ince
Volume of liquid from
micropipettes
Gravimetric evaluation
± 10%
±0.07% to 5.97%
Time
Compared to independent clock
± 2 seconds/hour
0 seconds/hour
Temperature
Compared to independent calibrated thermometer
±2 °C
± 0.1 to 0.2 °C
Relative Humidity
Compare to independent calibrated hygrometer
± 10%
± 0.3 to 1.7%
15
-------
3.3.2 Technical Systems Audit
Observations and findings from technical systems audits (TSAs) were documented and
submitted to the laboratory technical lead for response. TSAs were conducted on December 2
and 5, 2014, to ensure that tests were being conducted in accordance with the appropriate QAPP
and QMP. As part of the audit, test procedures were compared to those specified in the QAPP,
and data acquisition and handling procedures were reviewed. One deviation addressing the use
of sterile filtered water was noted during the TSA (see below).
3.3.3 Deviations
Two deviations occurred during this study. The first, a QAPP deviation, addressed the
inadvertent addition of language to the QAPP stating that sterile filtered water (SFW) would be
applied to the blank test coupons. These coupons were not nor were they intended to be
inoculated with SFW. The blank test coupons were used to determine background cytotoxicity
of each material type per test, and the addition of other materials such as SFW (if used) may
have changed cytotoxic effects.
The second deviation was prepared to address the failure of the iLOG software described in
Section 2.5. This failure occurred during Test 17, when a site-wide software update was applied
to the computer running the data collection software. Application of this update caused the
computer to restart, resulting in the loss of approximately one day of temperature and RH
readings. The impact of this deviation was considered minimal since the RH remained running
under operational conditions, and it takes at least 24 h for those to equilibrate after a cold start of
the system. Corrective actions were taken to remove the computer from the automatic update
list.
3.3.4 Data Quality Audit
At least 10% of the data acquired during the evaluation were audited. A QA auditor traced the
data from the initial acquisition, through reduction and statistical analysis, to final reporting to
ensure the integrity of the reported results. All calculations performed on the data undergoing the
audit were verified. Only minor issues were noted with the data, mostly manual data
transcription errors that were corrected.
3.4 QA/QC Reporting
Each assessment and audit was documented in accordance with the QAPP and QMP. For these
tests, findings were noted (none significant) in the data quality audit, and no follow-up corrective
action was necessary. The findings were mostly minor data transcription errors requiring some
recalculation of attenuation results, but none were gross errors in recording. QA/QC procedures
were performed in accordance with the QAPP.
3.5 Data Review
Records and data generated in the evaluation received a QC/technical review before they were
utilized in calculating or evaluating results and prior to incorporation in this report.
16
-------
4.0 Summary of Results and Discussion
The attenuation of purified and crude forms of ricin toxin inoculated onto porous and nonporous
material coupons was evaluated under various controlled environmental conditions and elapsed
times. For the eighteen tests in this evaluation, the environmental conditions ranged from 20-50
°C and 20-75% RH for durations of 6 h to 28 days. Tests 1 through 14 examined six different
material types while Tests 15 through 18 examined mild steel only, but with an increased number
of timed collection points at elevated temperatures. Test 18 included crude ricin only to further
increase the number of collection points evaluated.
4.1 Test Environmental Conditions
The temperature and RH during Tests 1-14 were controlled as described in Section 2.0.
Temperature and RH readings were taken once every minute for the duration of each test. The
actual environmental conditions for each test are shown in Table 4-1 and reported as the average
value ± standard deviation (SD). In Tests 15-18, temperature was controlled to either 40 or 50
°C, but RH was not controlled and averaged 20-27% RH. Actual temperatures were within ±1
°C of target, while RH was within ±3% of target RH.
Table 4-1. Actual Attenuation Conditions for Environmental Chamber Tests
Test
Target
Temperature
°C
Aetual
Temperature
°C
Target
%RH
Aetual
%RH
Contact Time (Days)
1
30
30.09±0.30
75
73.60±2.49
7
2
25
25.01±0.09
45
47.03±0.30
7
3
25
24.99±0.22
45
46.39±1.06
14
4
25
25.95±1.35
75
72.43±7.20
7
5
25
25.58±1.07
75
73.93±5.50
14
6
30
29.70±0.16
45
48.09±1.73
7
7
30
30.03±0.42
45
45.61±3.52
14
8
30
30.31±0.21
75
72.96±1.16
14
9
20
20.41±0.23
45
45.22±1.47
7
10
20
20.59±0.29
45
45.26±1.40
14
11
20
20.80±0.55
75
75.28±1.05
7
12
20
20.84±0.80
75
72.43±5.14
14
13
20
19.80±0.53
45
44.81±4.03
21
14
20
19.82±0.47
45
45.06±4.18
28
15
50
50.26±0.24
uncontrolled
21.05±2.67
6, 24, 30, 48, 72, and 96 h
16
40
39.95±0.43
uncontrolled
26.62±3.31
2, 3, 4, 5, 6, 7
17
50
50.41±0.72
uncontrolled
19.79±2.20
2, 3, 4, 5, 6, 7
18
40
40.37±0.49
uncontrolled
21.56±2.48
3,4, 5, 6, 7, 10, 11, 12, 13,
14
17
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4.2 Recovery of Ricin from Positive Controls
The average percent recoveries for the pure and crude ricin from the positive control test
materials are shown in Figure 4-1. These are the study-wide averages of the percent ricin
recovered one hour after the coupons were inoculated. The percent recoveries were calculated
based on a 250 |ig pure ricin inoculum and an average of 320 |ig inoculum of the crude form.
Average positive control recoveries by material ranged from 3 to 90% for pure ricin and 17 to
127% for the crude ricin. The positive control percent recoveries were generally higher for the
crude form, although the average recoveries were not significantly different for the two
preparations. Wood had the lowest average recovery at 3 and 17% for pure and crude ricin,
respectively, while carpet had the highest average recovery at 90 and 127% for pure and crude,
respectively. Note the wider variability (as standard deviation error bars) in recovery from the
crude ricin positive controls as compared to the pure ricin.
Average Percent Recovery of Pure and Crude Ricin from
Positive Controls ± SD
250%
200%
150% -
100%
: il ¦ ¦ . _ I .
Mild Steel Rubber Plastic Wood Carpet Paper
¦ Pure Ricin ¦ Crude Ricin
Figure 4-1. Summary of Average Percent Recovery from Positive Controls for Pure Ricin
and Crude Ricin by Material Type, ± Standard Deviation
4.3 Environmental Conditions Required for 99% Attenuation
Table 4-2 provides an overview of the study results in terms of presenting the few (seven) cases
where we observed >99% reduction on any of the material types tested (out of over 200 test
combinations of ricin type, temperature, RH, material, and contact time). As seen in Table 4-2, in
general, elevated temperature or RH was required to achieve greater than 99% reduction of pure
or crude ricin toxin. More specifically, there were no cases in which any form of ricin was
attenuated to a greater extent than 99% at 20 °C (either RH, up to 28 days) or at 25 °C/45% RH.
There was only one case where the crude ricin preparation was attenuated more than 99%.
Attenuation of more than 99% occurred most often on the mild steel and paper materials.
18
-------
The detailed attenuation results for each test and for each material are provided in Appendix A.
For each test and material, the results are presented in terms of the ricin recovered from each
replicate positive control and test coupon and the average percent reduction.
Table 4-2. Test Parameter Combinations Demonstrating Greater than 99% Reduction of
Ricin
Test*
Ricin
Form/Target
Mass
Temp
°C
%RH
Contact
Time
(Days)
Material
% Reduction
±CI
4
Pure/250 |ig
25 °C
75%
7
Mild Steel
99.87
0.11
Crude/320 |ig
7
Paper
99.54
0.12
5
Pure/250 |ig
25 °C
75%
14
Mild Steel
99.95
0.03
6
Pure/250 |ig
30 °C
45%
7
Pine
Wood
99.38
0.47
7
Paper
99.83
0.24
IT
Pure/250 |ig
50 °C
20%
6
Mild Steel
99.05
0.48
7
Mild Steel
99.92
0.02
* Detailed data from each test number can be referenced in Appendix A.
aThe only material tested was mild steel.
4.4 Attenuation Results for Tests at 20-30 °C
The tests conducted at 20-30 °C are representative of the environmental conditions that would be
expected to be achieved using the heating system of a building without any additional
equipment. For each of the tests conducted at 20-30 °C (1-14), percent reduction results by
material are shown in Figures 4-2 through 4-4. Pure and crude ricin extracts were recovered on
all material types tested for at least 28 days at 20 °C and 45% RH, and for at least 14 days for all
other environmental conditions tested. For crude ricin exposed to 20 °C/45% RH, recovery of the
toxin at 14 days was not significantly different from the positive control recoveries for most of
the materials (see Table la in Appendix B). Detailed values for the attenuation results for each
test are provided in Appendix A. See also Table 1 in Appendix B, which provides the detailed
numerical attenuation results by environmental condition, elapsed time, material, and ricin form.
Significant differences in attenuation between the two ricin forms, as a function of elapsed time,
or between environmental conditions on a specified material may be assessed visually via
Figures 4-2 through 4-4, i.e., depending on whether the 95% CIs of the two attenuation results
overlap. Generally, attenuation increased over time and at higher temperatures. However,
because there was a considerable amount of variability in the results (especially with the crude
ricin), in some cases attenuation may appear to have decreased over time or at higher
temperatures. Because of this variability, additional multiple statistical pair-wise comparisons of
the data were conducted using ANOVA models, the results of which are detailed in Section 4.6
and in Appendix B. These more robust statistical approaches allow us to better assess the effect
19
-------
of the environmental conditions and materials on attenuation, and these results are summarized
in Section 4.6.
20
-------
Percent Reduction of Ricin on Mild Steel
120.00
u 100.00
+i
o 80.00
-M
(J
60.00
(D
CC
c 40.00
(D
U
&_
a! 20.00
0.00
120.00
~ 100.00
+1
c
o
u
D
"D
a»
cc
+-»
c
cu
u
&_
a;
Q_
80.00
60.00
40.00
20.00
0.00
30°C 45% 30°C 70%
20°C 45% 20°C 75% 25°C45% 25°C 75%
Note: Tests were carried out to 21 and 28 days only for 20°C and 45% RH
¦ Pure Day 7 ¦ Pure Day 14 I Pure Day 21 ¦ Pure Day 28
¦ Crude Day 7 ¦ Crude Day 14 Crude Day 21 ¦ Crude Day 28
Percent Reduction of Ricin on Rubber
I
20°C 45% 20°C 75% 25°C45% 25°C 75% 30°C45% 30°C 70%
Note: Tests were carried out to 21 and 28 days only for 20°C and 45% RH
¦ Pure Day 7 ¦ Pure Day 14 ¦ Pure Day 21 ¦ Pure Day 28
¦ Crude Day 7 ¦ Crude Day 14 ¦ Crude Day 21 ¦ Crude Day 28
Figure 4-2. Summary of Percent Reduction (Tests 1-14) Results for Steel and Rubber, by
Environmental Condition, Comparing Pure and Crude Ricin ± 95% Confidence Interval
21
-------
Percent Reduction of Ricin on Plastic
120.00
_ 100.00
u
+1
c
o
80.00
u
D
~a
a»
en
60.00
g 40.00
a)
CL
20.00
0.00
120.00
_ 100.00
u
+i
c
o
80.00
o
60.00
(D
CC
| 40.00
a»
o_
20.00
0.00
T
l
20°C 45% 20°C 75% 25°C45% 25°C 75%
Note: Tests were carried out to 21 and 28 days only for 20°C and 45% RH
30°C 45% 30°C 70%
I Pure Day 7 ¦ Pure Day 14 I Pure Day 21 ¦ Pure Day 28
I Crude Day 7 ¦ Crude Day 14 Crude Day 21 ¦ Crude Day 28
Percent Reduction of Ricin on Wood
20°C 45% 20°C 75% 25°C45% 25°C 75% 30°C45% 30°C 70%
Note: Tests were carried out to 21 and 28 days only for 20 °C and 45% RH
¦ Pure Day 7 ¦ Pure Day 14 ¦ Pure Day 21 ¦ Pure Day 28
¦ Crude Day 7 ¦ Crude Day 14 ¦ Crude Day 21 ¦ Crude Day 28
Figure 4-3. Summary of Percent Reduction (Tests 1-14) Results for Plastic and Wood, by
Environmental Condition, Comparing Pure and Crude Ricin ± 95% Confidence Interval
22
-------
120.00
_ 100.00
u
+i
c
o
80.00
u
¦a
a)
oc
60.00
S 40.00
a)
CL
20.00
0.00
120.00
Percent Reduction of Ricin on Carpet
1
20°C 45% 20°C 75% 25°C45% 25°C 75% 30°C45% 30°C 70%
Note: Tests were carried out to 21 and 28 days only for 20 °C and 45% RH
¦ Pure Day 7 ¦ Pure Day 14 ¦ Pure Day 21 ¦ Pure Day 28
¦ Crude Day 7 ¦ Crude Day 14 ¦ Crude Day 21 ¦ Crude Day 28
Percent Reduction of Ricin on Paper
100.00
80.00
60.00
40.00
20.00
20°C 45% 20°C 75% 25°C45% 25°C 75% 30°C45% 30°C 70%
Note: Tests were carried out to 21 and 28 days only for 20 °C and 45% RH
¦ Pure Day 7 ¦ Pure Day 14 ¦ Pure Day 21 1 Pure Day 28
¦ Crude Day 7 ¦ Crude Day 14 ¦ Crude Day 21 ¦ Crude Day 28
Figure 4-4. Summary of Percent Reduction (Tests 1-14) Results on Carpet and Paper, by
Environmental Condition, Comparing Pure and Crude Ricin ± 95% Confidence Interval
23
-------
In Table 4-3, we provide a summary of the attenuation results, by environmental condition, for
the tests conducted at 20-30 °C. To allow for simple comparisons, the percent reduction results
were averaged across all materials for the 14-day elapsed time period, since all six environmental
conditions were evaluated at this time point. Fourteen days was the longest test duration
investigated for the study, with the exception of the 20 °C/45% RH condition, which was tested
out to 28 days. The attenuation data for 20 °C/45% RH condition at 28 days are also included in
Table 4-3 for comparison purposes. While Table 4-3 provides a quick snapshot of results and
illustrates some of the confounding effects of test parameters, as mentioned above, Section 4.6
provides a detailed discussion of the statistical assessment of significant effects of test variables.
Table 4-3. Average Percent Attenuation Obtained for Each Environmental Condition
at 14 and 28 days
Temp
°C/%RH
Test duration
(days)
Average %
Attenuation
for Pure
Riein*
Average %
Attenuation
for Crude
Riein*
20/45
14
63%
7%
20/75
14
58%
56%
25/45
14
88%
73%
25/75
14
88%
51%
30/45
14
75%
81%
30/75
14
63%
39%
20/45
28
80%
77%
* Average of all six materials
4.5 Attenuation Results for Tests at 40-50 °C
The tests conducted at the elevated temperatures of 40 and 50 °C are representative of
environmental conditions that could be achieved in a structure with additional, ancillary heating
equipment. Percent reduction results for mild steel only, for each test (15-18), are shown in
Figures 4-5 through 4-8. Steel was selected for these tests based on its lower variability in
attenuation results (e.g., as exhibited in Figures 4-2 to 4-4). The pure ricin shows steady
degradation over time, while the attenuation of the crude ricin over time is more subdued. Over
90% reduction of the pure ricin occurs within two days at both 40 and 50 °C, with 99% reduction
obtained in six days at 50 °C (Figure 4-7). For the crude ricin on mild steel, 98% attenuation
occurred after one week at 50 °C. In test 18, where we focused only on crude ricin on mild steel
to allow for multiple test durations for up to two weeks, the maximum attenuation obtained was
only 79%) at 40 °C (Figure 4-8). Detailed values for the attenuation results are provided in
Appendix A.
24
-------
Mild Steel Percent Reduction 50°CTest 15
120.00
u
+i
100.00
80.00
-a 60.00
Cl)
OC
| 40.00
20.00
illLLL
6 Hrs 24 Hrs 30 Hrs 48 Hrs 72 Hrs 96Hrs
¦ Pure ¦ Crude
Figure 4-5. Summary of Percent Reduction (Test 15) Results at 50 °C, by Time,
Comparing Pure and Crude Ricin ± 95% Confidence Interval; RH uncontrolled
0.00
Mild Steel Percent Reduction 40°C Test 16
LLILLL
2 Days 3 Days 4 Days 5 Days 6 Days 7 Days
¦ Pure ¦ Crude
Figure 4-6. Summary of Percent Reduction (Test 16) Results at 40 °C, by Day,
Comparing Pure and Crude Ricin ±95% Confidence Interval; RH uncontrolled
120.00
M 100.00
u
e 80.00
o
o
-o 60.00
§ 40.00
(L)
20.00
0.00
25
-------
Mild Steel Percent Reduction 50°CTest 17
120
100
u
+i
c
o
4-»
o
~o
<1)
QC
4-»
c
CD
u
1_
(D
CL
80
60
40
20
48 Hrs 72 Hrs 96 Hrs 120 Hrs
¦ Pure ¦ Crude
144 Hrs
168 Hrs
Figure 4-7. Summary of Percent Reduction (Test 17) Results, by Time, Comparing Pure
and Crude Ricin ± 95% Confidence Interval; RH uncontrolled
120.00
100.00
u
+i
c
o
*+-»
(J
D
~a
cu
cc:
80.00
60.00
S 40.00
o
0)
Q_
20.00
0.00
Mild Steel Percent Reduction 40°CTest 18
3 Days 4 Days 5 Days 6 Days 7 Days 10 Daysll Daysl2 Daysl3 Daysl4 Days
¦ Crude
Figure 4-8. Summary of Percent Reduction (Tests 18) Results at 40 °C, by Day, for Crude
Ricin ± 95% Confidence Interval; RH uncontrolled
26
-------
4.6 Statistical Analyses to Assess Effect of Test Variables on Attenuation
Effect of elapsed time
The results of statistical analyses indicating whether the ricin recoveries over time were significantly
different from the ricin recovered at TO (time zero, positive control recoveries) are shown in Table 1
of Appendix B. The results for additional pair-wise comparisons for all other time points for a given
experimental condition and material are shown in Table 3 of Appendix B. Ricin was generally more
stable at the lower temperatures, and increasing elapsed times at the lower temperatures in many
cases did not improve attenuation. This effect was more pronounced with the crude ricin. For
example, there was generally no significant attenuation of crude ricin up to 14 days at 20 °C/45% RH
(Table 1A of Appendix B). At this same environmental condition, minor but statistically significant
attenuation occurred for the pure ricin at 7 days on most of the materials. Tables 3a, 3b, and 3c of
Appendix B also illustrate this effect. Even at the higher temperature of 40 °C, there was no
significant attenuation of the crude ricin at five days (Table 1H of Appendix B). Alternatively, there
was also the case in which no significant additional attenuation occurred with each successive time
point if the attenuation was already relatively high. See, for example, the pure ricin attenuation over
time at 50 °C shown in Figure 4-7.
Effect of temperature
The statistical results of the pair-wise comparisons to assess the effect of temperature on the
attenuation of ricin are shown in Appendix B, Tables 6 and 7 (refer to Tukey-adjusted P-values). In
the majority of cases, there was either no significant change in attenuation or there was an increase in
attenuation associated with increasing temperature. Very few comparisons showed that the
attenuation decreased with increasing temperature. The effect of increasing temperature on
attenuation was more pronounced when comparing results with a 10 °C temperature difference (e.g.,
attenuation at 20 versus 30 °C).
Effect of material
The results of the nearly 500 pair-wise comparisons between materials are shown in Table 2 of
Appendix B. (Refer to Tukey-adjusted P-values; each material was compared a total of 140 times.) In
the majority of the comparisons, there was no significant difference in attenuation between materials.
However, the mild steel material did have the largest number of comparisons in which ricin was
attenuated to a significantly greater extent than the other material being compared. The ranking of
materials by the number of comparisons where ricin was attenuated to a significantly greater extent
than the material it was compared with (shown in parentheses) is as follows: steel (58), paper (30),
carpet (24), plastic (12), rubber (7) and wood (2). According to this ranking, ricin was least
attenuated on the wood material.
Effect of RH
With all other factors being equivalent, increasing the RH from 45 to 75% does not appear to affect
attenuation. As Table 5 of Appendix B shows, in over half the pair-wise comparisons, there was no
significant effect on attenuation when increasing the RH (refer to Tukey-adjusted P-values). Further,
in the 33 cases where there was a significant effect of increasing the RH from 45 to 75%, 16 of the
cases showed an increase in attenuation while 17 of the cases showed a decrease in attenuation. The
effect of RH (or lack thereof) was similar for both ricin types. However, the effect of RH appears to
be somewhat dependent on the material. For example, for both mild steel and wood materials, there
27
-------
was either no effect of RH, or an increase in RH significantly increased the ricin attenuation in nearly
all of the comparisons. The opposite effect occurred with the plastic and rubber materials.
Effect of Ricin Preparation
A summary of the attenuation results, comparing the average percent reduction ± SD for the pure
and crude ricin, by material, is shown in Figure 4-9. These results are averages for Tests 1-14, in
which all materials were subject to the same environmental conditions. Overall, the average
percent reductions by material ranged from 46.4 to 66.8% for crude ricin and 38.4 to 93.5% for
pure ricin. The pure ricin on mild steel, neoprene rubber, optical plastic, and wood exhibited a
higher percent reduction as compared to the crude material. The average attenuation of both
ricin types was highest on the steel material, although this effect was significant only for the pure
form.
Similar to the positive control recovery results, these attenuation results also highlight the higher
variability of the crude ricin as compared to the pure ricin. This variability is most likely due to
the potential for interactions of other proteins in solution that were not removed as they would be
in the commercially available pure material. Additionally, while the crude ricin suspension
constituents likely increased variability, they may also have shielded or protected the ricin
proteins from the environmental conditions, as evidenced by the increased stability.
Tables 4a-4h of Appendix B show the p-values associated with each pair-wise comparison to
assess the effect of the ricin preparation on attenuation and indicate the comparisons where there
was a significant difference (refer to Tukey-adjusted P-values). The comparison of percent
reduction by ricin type is presented for each material, environmental condition, and test duration.
The majority of comparisons show that there was either no significant effect of ricin type, or that
the crude preparation was attenuated significantly less. There were only a few cases in which the
crude ricin preparation was attenuated significantly more than the pure. Interestingly, the tests at
the elevated temperatures of 40 and 50 °C and with mild steel allowed for significant differences
in the two types of ricin to become more readily apparent. With these higher temperatures, the
crude form was significantly less attenuated than the pure form at all but two time points (Tables
4g and 4h of Appendix B).
28
-------
Pure vs. Crude Ricin Percent Reduction
¦ Pure Ricin ¦ Crude Ricin
Figure 4-9. Comparison of Average Percent Reduction for Pure Ricin and Crude Ricin by
Material Type ± Standard Deviation, for Tests 1-14 (20-30 °C)
4.7 Summary
Over the entire study, there were only seven cases (out of over 200 test combinations of ricin
type, temperature, RH, material, and contact time) in which we observed greater than 99%
attenuation of ricin. In general, elevated temperature or RH was required to achieve greater than
99% reduction of pure or crude ricin toxin. More specifically, there were no cases where any
form of ricin was attenuated to a greater extent than 99% at 20 °C or at 25 °C/45% RH. There
was only one case where the crude ricin preparation was attenuated more than 99%. Attenuation
of more than 99% occurred most often on the mild steel and paper materials.
Under the environmental conditions most resembling the indoor environment, significant
attenuation of the crude ricin was not observed until at least 21 days, with the exception of mild
steel. At 30 °C, the average ricin attenuation across all materials and ricin forms ranged from 39-
81%. Per the statistical analyses, there was either no significant change in attenuation, or there
was an increase in attenuation associated with increasing temperature. The effect of increasing
temperature on attenuation was more pronounced when comparing results with a 10 °C
temperature difference.
For the pure ricin, heat treatments at the elevated temperatures of 40 °C for 5 days and 50 °C for
2-3 days achieved greater than 96% attenuation. For the crude ricin preparation, appreciable
recovery of the ricin still occurred at 40 °C after two weeks. A seven-day heat treatment at 50 °C
was required to achieve greater than 98% attenuation of the crude ricin on mild steel.
29
-------
In general, increasing the RH level from 45 to 75% did not significantly affect attenuation of ricin.
The effect of RH (or lack thereof) was similar for both ricin types, while the effect of RH appeared to
be somewhat dependent on the material. For example, for both the mild steel and wood materials,
there was either no effect of RH, or an increase in RH significantly increased the ricin attenuation in
nearly all of the comparisons. The opposite effect of RH occurred with the plastic and rubber
materials.
In the majority of the statistical comparisons, there was no significant difference in attenuation
between the materials. However, the mild steel material did have the largest number of comparisons
in which ricin was attenuated significantly more than the other material being compared. The ranking
of materials by the number of comparisons in which ricin was attenuated significantly more than the
material it was compared with (shown in parentheses) is as follows: steel (58), paper (30), carpet
(24), plastic (12), rubber (7) and wood (2).
In the majority of tests, the crude ricin was more stable, i.e., more difficult to attenuate, than the
pure ricin. The attenuation results for the crude ricin also exhibited more variability than the pure
ricin. The use of a biological system, i.e., a cell-based assay, to quantitate ricin toxicity,
regardless of ricin type, may also have contributed to variability in results. The sometimes high
variability in results may mask the effects of test variables, and so a robust statistical analysis
was conducted. From this statistical analysis, we found that the crude ricin was attenuated
significantly more than the pure form in only a few cases. In other words, in the overwhelming
majority of the statistical comparisons made, there was either no significant effect of ricin
preparation on attenuation, or the pure ricin was attenuated significantly more than the crude
form. The tests on mild steel at the elevated temperatures of 40 and 50 °C allow for significant
differences between the two ricin forms to become more readily apparent, i.e., the pure ricin
form is more easily attenuated.
30
-------
5.0 References
1. US Centers for Disease Control and Prevention. 2009. Biosafety in Microbiological and
Biomedical Laboratories. 5th Edition, HHS Publication No. (CDC) 21-1112.
2. US EPA, 2006. Impact of Temperature and Humidity on the Persistence of Vaccinia Virus
and Ricin Toxin on Indoor Surfaces. US EPA Report 600/R-08/002.
3. US Army Medical Research Institute of infectious Diseases. 2005. Medical Management of
Biological Casualties Handbook, Sixth Edition.
4. Barnes DJ, Baldwin BS, Braasch DA. 2009. Degradation of Ricin in Castor Seed Meal by
Temperature and Chemical Treatment. Industrial Crops and Products (29): 509-515
5. Anandan S, Anil Kumar GK, Ghosh J, Ramachandra KS. 2005. Effects of Different Physical
and Chemical Treatments on Detoxification of Ricin in Castor Cake. Animal Feed Science
and Technology (120): 159-168
6. US EPA, 2005. Workshop on Decontamination, Cleanup, and Associated Issues for Sites
Contaminated with Chemical, Biological, or Radiological Materials. US EPA Report
EPA/600/R-05/083.
7. US EPA. 2014. Quality Assurance Project Plan for the Neutralization of Ricin Toxicity
Using Elevated Temperature and Humidity, Version 1. June. (Available upon request by
contacting EPA).
8. US EPA, 2015. Evaluation of the Inactivation of Ricin Toxin on Surfaces Using Vapor Phase
Hydrogen Peroxide. US EPA Report 600/R-15/131.
9. Lin T T-S and Li S S-L. 1980. Purification and Physicochemical Properties of Ricins and
Agglutinins from Ricinus communis. European Journal of Biochemistry (105):453-459.
10. US EPA. 2013. Evaluation of Ethylene Oxide for the Inactivation of Bacillus anthracis. US
EPA Technology Evaluation Report. EPA/13/R-13-220.
11. Mosmann, T. 1983. Rapid Colorimetric Assay for Cellular Growth and Survival: Application
to Proliferation and Cytotoxicity Assays." Journal of Immunological Methods (65): p. 55-63.
31
-------
Appendix A
Detailed Test Results
Attenuation Results
The detailed attenuation results for varied environmental conditions against pure and crude ricin toxin on six
material types (mild steel, rubber, optical plastic, carpet, bare pine wood and paper) are shown in Tables A-l
through A-3. Data highlighted green indicate >99 % reduction.
Table A-l. Attenuation of Pure Ricin Toxin"
Test
Test Parameters
Material
Inoculum
Mean Recovered Ricin ± SD
(|ig coupon)
" oReduction
Number
Temp
°C±SD
%RH
±SD
Time
(ug coupon)
Positive Control
Test Coupon
±CI
Mild Steel
120.170 ±77.058
8.140 ±4.781
93.23 ±5.16
Neoprene Rubber
41.843 ±65.194
10.764 ±3.524
74.27 ±35.90
1
30.09 ±
73.60 ±
7 Days
Optical Plastic
250
87.882 ± 79.708
3.753 ±0.580
95.73 ±3.44
0.30
2.49
Bare Pine Wood
Industrial Carpet
Paper
9.536 ± 1.760
241.153 ±22.577
95.309 ±34.061
3.050 ± 1.148
65.399 ±49.019
5.052 ± 1.162
68.02 ± 11.75
72.88 ± 17.96
94.70 ± 1.97
Mild Steel
180.510 ±38.728
20.703 ± 3.678
88.53 ±2.80
Neoprene Rubber
56.103 ±34.557
51.101 ±24.325
8.92 ±62.15
25.01 ±
47.03 ±
7 Days
Optical Plastic
250
198.226 ±80.640
34.049 ± 3.400
82.82 ±6.31
0.09
0.30
Bare Pine Wood
Industrial Carpet
Paper
5.377 ±2.234
154.571 ±91.412
142.943 ±59.814
7.655 ±2.751
87.719 ± 14.440
65.853 ±39.590
f0.00± 68.55
43.25 ±30.54
53.93 ±29.58
Mild Steel
180.510 ±38.728
6.380 ±2.652
96.47 ± 1.45
Neoprene Rubber
56.103 ±34.557
10.384 ±4.298
81.49 ± 12.04
3
24.99 ±
0.22
46.39 ±
1.06
14 Days
Optical Plastic
Bare Pine Wood
Industrial Carpet
Paper
250
198.226 ±80.640
5.377 ±2.234
154.571 ±91.412
142.943 ±59.814
8.358 ±2.051
2.081 ± 1.011
7.678 ±3.542
8.147 ±5.807
95.78 ± 1.76
61.30 ±21.68
95.03 ±3.27
94.30 ±4.13
Mild Steel
68.378 ±49.618
0.089 ±0.051
99.87 ± 0.11
Neoprene Rubber
149.260 ± 24.963
34.536 ± 11.134
76.86 ±7.37
25.95 ±
72.43 ±
7 Days
Optical Plastic
250
50.373 ±60.376
38.419 ±7.183
23.73 ±81.10
1.35
7.20
Bare Pine Wood
Industrial Carpet
Paper
12.774 ±9.959
206.655 ±38.789
60.340 ±39.655
4.171 ± 1.793
83.698 ± 58.006
20.231 ±4.503
67.35 ± 25.48
59.50 ±25.49
66.47 ±20.39
Mild Steel
68.378 ±49.618
0.033 ±0.011
99.95 ±0.03
Neoprene Rubber
149.260 ± 24.963
11.959 ±6.604
91.99 ±4.05
5
25.58 ±
1.07
73.93 ±
5.50
14 Days
Optical Plastic
Bare Pine Wood
Industrial Carpet
Paper
250
50.373 ±60.376
12.774 ±9.959
206.655 ±38.789
60.340 ±39.655
20.927 ± 1.561
1.759 ± 1.309
15.097 ± 10.671
3.403 ± 1.829
58.46 ± 43.73
86.23 ± 13.01
92.69 ±4.68
94.36 ±4.20
3 Data are expressed as the mean (± SD) of the mass of toxin recovered on five replicate individual samples, and attenuation (percent reduction ± CI).
b Positive Controls = samples inoculated, not attenuated (recovered after 1 hour).
c Test Coupons = samples inoculated, attenuated.
d CI = confidence interval (± 1.96 x standard error [SE]).
e % Reduction calculated as (mean ricin recovered positive controls - mean ricin recovered test coupons)/ mean ricin recovered positive controls
f As a result of high variability, negative value reported as "0".
A-l
-------
Table A-l. Attenuation of Pure Ricin Toxin" (Continued)
Test
Number
Test Parameters
Material
Inoculum
(ug coupon)
Mean Recovered Ricin ± SD
(|ig coupon)
" oReduction ±
Temp °C
%RH
Time
Positive Control
Test Coupon
CI
Mild Steel
159.998 ±26.952
25.250 ±4.137
84.22 ±3.25
Neoprene Rubber
167.446 ±40.218
20.872 ± 19.731
87.54 ± 10.66
29.70 ±
48.09 ±
7 Days
Optical Plastic
250
232.860 ±27.712
49.522 ±4.462
78.73 ±2.78
0.16
1.73
Bare Pine Wood
5.396 ± 1.198
0.694 ±0.411
87.14 ± 7.13
Industrial Carpet
204.026 ± 98.436
1.262 ±0.914 1
99.38 ±0.47
Paper
56.780 ±57.697
0.094 ±0.121
99.83 ±0.24
Mild Steel
159.998 ±26.952
13.685 ±5.835
91.45 ±3.44
Neoprene Rubber
167.446 ±40.218
31.055 ±6.671
81.46 ±5.24
7
30.03 ±
0.42
45.61 ±
3.52
14 Days
Optical Plastic
Bare Pine Wood
Industrial Carpet
Paper
250
232.860 ±27.712
5.396 ± 1.198
204.026 ± 98.436
56.780 ±57.697
21.643 ±9.633
3.808 ±0.775
80.311 ± 18.555
4.630 ±3.097
90.71 ±3.75
29.44 ± 18.63
60.64 ± 18.46
91.85 ±8.69
Mild Steel
90.506 ±41.338
4.557 ± 10.096
94.97 ±9.98
Neoprene Rubber
56.642 ± 18.047
22.361 ±2.860
60.52 ± 11.88
30.31 ±
72.96 ±
14 Days
Optical Plastic
250
49.017 ± 16.041
14.399 ±3.614
70.62 ± 10.62
0.21
1.16
Bare Pine Wood
Industrial Carpet
Paper
1.938 ± 1.531
147.407 ±89.460
59.617 ±60.120
6.836 ±2.793
44.416 ±20.943
14.708 ± 2.498
f0.00 ± 274.96
69.87 ±20.30
75.33 ±22.11
Mild Steel
192.891 ±37.467
48.573 ± 15.609
74.82 ±8.29
Neoprene Rubber
114.993 ±36.407
101.073 ±9.998
12.11 ±25.55
20.41 ±
45.22 ±
7 Days
Optical Plastic
250
199.895 ±48.331
24.371 ±31.664
87.81 ± 14.12
0.23
1.47
Bare Pine Wood
Industrial Carpet
Paper
13.015 ±3.962
206.481 ±35.515
138.476 ± 13.787
19.645 ± 15.955
126.246 ±69.684
80.794 ±36.946
0.00 ± 114.75
38.86 ±30.98
41.65 ±23.93
Mild Steel
192.891 ±37.467
15.611 ±22.539
91.91 ± 10.33
Neoprene Rubber
114.993 ±36.407
44.091 ± 11.210
61.66 ± 13.65
10
20.59 ±
0.29
45.26 ±
1.40
14 Days
Optical Plastic
Bare Pine Wood
Industrial Carpet
Paper
250
199.895 ±48.331
13.015 ±3.962
206.481 ±35.515
138.476 ± 13.787
43.656 ± 10.448
31.785 ±24.921
14.047 ±7.134
79.301 ±24.979
78.16 ±6.51
0.00 ± 180.04
93.20 ±3.20
42.73 ± 16.58
Mild Steel
224.689 ±96.111
5.056 ±0.931
97.75 ±0.92
Neoprene Rubber
98.829 ± 19.853
86.337 ±7.451
12.64 ± 16.74
11
20.80 ±
75.28 ±
7 Days
Optical Plastic
250
78.392 ± 8.647
89.316 ± 15.869
0.00 ±20.89
0.55
1.05
Bare Pine Wood
Industrial Carpet
Paper
2.569 ±0.855
291.579 ±49.341
90.726 ± 57.043
11.335 ±5.337
208.112 ±41.463
86.250 ±9.367
0.00 ± 222.93
28.63 ± 16.35
4.93 ±53.17
Mild Steel
224.689 ±96.111
5.313 ±5.312
97.64 ±2.25
Neoprene Rubber
98.829 ± 19.853
34.733 ±6.186
64.86 ±8.27
12
20.84 ±
0.80
72.43 ±
5.14
14 Days
Optical Plastic
Bare Pine Wood
Industrial Carpet
Paper
250
78.392 ± 8.647
2.569 ±0.855
291.579 ±49.341
90.726 ± 57.043
32.356 ± 15.284
3.913 ±0.580
107.434 ±30.672
40.351 ± 14.248
58.73 ± 17.55
0.00 ±48.62
63.15 ± 10.72
55.52 ±28.11
Mild Steel
192.38 ±40.482
16.393 ±7.916
91.48 ±3.93
Neoprene Rubber
203.877 ±26.250
28.306 ± 5.162
86.12 ±2.72
13
19.80 ±
0.53
44.81 ±
4.03
21 Days
Optical Plastic
Bare Pine Wood
Industrial Carpet
Paper
250
214.895 ±33.296
4.706 ± 1.723
349.205 ± 43.469
82.853 ±23.078
27.605 ± 2.390
1.803 ± 1.039
91.676 ±27.681
54.022 ±20.863
87.15 ±2.00
61.68 ±22.93
73.75 ±7.52
47.48 ± 20.56
3 Data are expressed as the mean (± SD) of the mass of toxin recovered on five replicate individual samples, and attenuation (percent reduction ± CI).
b Positive Controls = samples inoculated, not attenuated.
c Test Coupons = samples inoculated, attenuated.
d CI = confidence interval (± 1.96 x standard error [SE]).
f As a result of high variability, negative value reported as "0".
A-2
-------
Table A-l. Attenuation of Pure Ricin Toxin" (Continued)
lost
Tost Parameters
Material
Inoculum
Mean Recovered Ricin ± SD
(|ig coupon)
"oReduclion
X Limber
Temp :C
° oR 11
Time
(ug coupon)
Positive Control
Test Coupon
CI
Mild Steel
192.38 40.482
2.265 1.447
98.82 0.69
Neoprene Rubber
203.877 + 26.250
22.566 + 6.892
88.93 + 3.22
14
19.82 ±
0.47
45.06 ±
4.18
28 Days
Optical Plastic
Bare Pine Wood
Industrial Carpet
Paper
250
214.895 + 33.296
4.706+ 1.723
349.205 ± 43.469
102.853 + 23.078
18.761 + 7.392
5.428 + 4.933
44.936 ± 47.379
34.173 + 12.943
91.27 + 3.24
0.00 + 99.06
87.13+ 11.98
66.77+ 12.82
Mild Steel TO
263.835 + 27.709
NA
NA
Mild Steel +6 h
97.816+ 17.229
62.93 + 6.66
15
50.26 ±
0.24
21.05 ±
2.67
Multiple
Mild Steel +24 h
Mild Steel +30 h
Mild Steel +48 h
Mild Steel +72 h
Mild Steel +96 h
250
NAg
37.059 + 3.819
23.953 + 2.656
19.739 + 6.912
5.002 + 0.972
3.546 + 0.834
85.95 + 1.81
90.92+ 1.22
92.52 + 2.40
98.10 + 0.37
98.66 + 0.30
Mild Steel TO
182.500 + 32.073
NA
NA
Mild Steel +48 h
17.684 + 3.793
90.31 + 2.36
39.95 ±
0.43
26.62
±3.31
Mild Steel +72 h
12.289 + 3.437
93.27+ 1.95
16
Multiple
Mild Steel +96 h
Mild Steel +120 h
Mild Steel +144 h
Mild Steel +168 h
250
NA
9.697 + 2.267
5.913 + 1.685
5.516 + 0.753
6.180 + 3.056
94.69+ 1.36
96.76 + 0.95
96.98 + 0.59
96.61 ± 1.56
Mild Steel TO
200.912 + 42.567
NA
NA
Mild Steel +48 h
5.713 + 1.310
97.16+ .078
17
50.41 ±
0.72
19.79 ±
2.20
Multiple
Mild Steel +72 h
Mild Steel +96 h
Mild Steel +120 h
Mild Steel +144 h
Mild Steel +168 h
250
NA
4.023 + 1.167
3.102 + 0.974
2.147+ 1.028
1.902+ 1.018
0.153 + 0.042
98.00 + 0.63
98.46 + 0.51
98.93 + 0.49
99.05 + 0.48
99.92 + 0.02
3 Data are expressed as the mean (± SD) of the mass of toxin recovered on five replicate individual samples, and attenuation (percent reduction ± CI).
b Positive Controls = samples inoculated, not attenuated.
c Test Coupons = samples inoculated, attenuated.
d CI = confidence interval (± 1.96 x standard error [SE]).
f As a result of high variability, negative value reported as "0".
§NA = Not applicable.
A-3
-------
Table A-2. Attenuation of Crude Ricin Toxin"
Test
Number
Test Parameters
Material
Inoculum
(ug coupon)
Mean Recovered Ricin ± SD
(|ig coupon)
" oReduction ±
Temp °C
%RH
Time
Positive Control
Test Coupon
CI
Mild Steel
215.692 ±83.217
3.748 ± 1.744
98.26 ±0.92
Neoprene Rubber
303.449 ± 135.904
213.755 ±48.948
29.56 ±31.06
1
30.09 ±
0.30
73.60 ±
2.49
7 Days
Optical Plastic
Bare Pine Wood
Industrial Carpet
Paper
320
297.116 ±75.901
50.730 ± 30.423
331.333 ± 107.507
225.850 ±334.821
147.589 ±78.831
25.121 ±10.499
323.101 ±57.961
12.990 ±2.421
50.33 ±25.78
50.48 ±31.73
2.48 ±31.69
94.25 ±7.53
Mild Steel
174.907 ±29.476
104.933 ± 10.086
40.01 ± 10.20
Neoprene Rubber
383.491 ±25.588
221.305 ± 116.120
42.29 ± 26.76
2
25.01 ±
0.09
47.03 ±
0.30
7 Days
Optical Plastic
Bare Pine Wood
Industrial Carpet
Paper
320
378.131 ±232.700
52.002 ± 26.692
278.805 ±234.893
467.700 ± 99.403
182.085 ± 50.571
29.364 ± 13.249
361.882 ±65.973
64.740 ± 87.050
51.85 ±28.50
43.53 ±33.83
f0.00 ± 98.07
86.16 ± 16.52
Mild Steel
174.907 ±29.476
114.113 ±42.250
34.76 ± 24.64
Neoprene Rubber
383.491 ±25.588
44.519 ± 8.830
88.39 ±2.13
3
24.99 ±
0.22
46.39 ±
1.06
14 Days
Optical Plastic
Bare Pine Wood
Industrial Carpet
Paper
320
378.131 ±232.700
52.002 ± 26.692
278.805 ±234.893
467.700 ± 99.403
150.286 ±99.070
24.654 ± 16.321
73.737 ±57.494
57.613 ±68.103
60.26 ±31.42
52.59 ±34.81
73.55 ±26.61
87.68 ± 12.97
Mild Steel
13.244 ± 13.139
2.330 ±0.890
82.41 ± 16.39
Neoprene Rubber
217.184 ±250.392
226.775 ± 139.189
f0.00± 119.54
25.95 ±
72.43 ±
7 Days
Optical Plastic
320
18.129 ± 11.914
5.423 ±7.397
70.08 ± 39.70
1.35
7.20
Bare Pine Wood
Industrial Carpet
10.353 ±4.519
97.020 ± 178.789
12.848 ± 15.967
45.245 ±95.995
f0.00 ±143.28
53.37 ± 114.87
Paper
335.223 ±94.148
1.557 ±0.068 |
99.54 ± 0.12
Mild Steel
13.244 ± 13.139
0.511 ±0.089
96.14 ± 3.41
Neoprene Rubber
217.184 ±250.392
209.061 ±94.556
3.74 ± 104.49
5
25.58 ±
1.07
73.93 ±
5.50
14 Days
Optical Plastic
Bare Pine Wood
Industrial Carpet
Paper
320
18.129 ± 11.914
10.353 ±4.519
97.020 ± 178.789
335.223 ±94.148
66.174 ±25.146
16.577 ± 12.620
2.274 ±0.577
12.283 ±4.807
f0.00± 242.88
f0.00± 123.17
97.66 ±3.82
96.34 ± 1.55
3 Data are expressed as the mean (± SD) of the mass of toxin recovered on five replicate individual samples, and attenuation (percent reduction ± CI).
b Positive Controls = samples inoculated, not attenuated.
c Test Coupons = samples inoculated, attenuated.
d CI = confidence interval (± 1.96 x standard error [SE]).
f As a result of high variability, negative value reported as "0".
A-4
-------
Table A-2. Attenuation of Crude Ricin Toxin" (Continued)
Tosl
Tosl Parameters
Material
Inoculum
Mean Recovered Ricin ± SD
(|ig coupon)
"^Reduction
Number
Temp C
° oR 11 T ime
(ug coupon)
Positive Control
Test Coupon
CI
Mild Steel
258.204 50.424
124.811 83.041
51.66 29.38
Neoprene Rubber
510.325 ± 107.271
59.947 ±62.351
88.25 ± 10.93
29.70 ±
48.09 ±
1.73 7DayS
Optical Plastic
320
320.984 ±94.865
12.334 ± 14.107
96.16 ±3.98
6 0.16
Bare Pine Wood
49.605 ± 17.156
44.749 ±31.096
9.79 ±61.38
Industrial Carpet
625.155 ± 137.038
172.604 ±222.875
72.39 ±31.70
Paper
33.410 ±23.788
207.509 ± 181.400
f0.00± 613.79
Mild Steel
258.204 ±50.424
43.730 ±41.418
83.06 ± 14.36
Neoprene Rubber
510.325 ± 107.271
188.890 ±202.132
62.99 ±35.38
30.03 ±
0.42
45.61 ±
3.52 14DayS
Optical Plastic
Bare Pine Wood
320
320.984 ±94.865
49.605 ± 17.156
70.771 ± 39.473
16.510 ±7.833
77.95 ± 12.20
66.72 ± 17.13
Industrial Carpet
625.155 ± 137.038
39.462 ± 19.850
93.69 ±3.04
Paper
33.410 ±23.788
25.413 ±28.179
23.94 ± 87.86
Mild Steel
99.014 ±58.918
6.000 ±2.856
93.94 ±4.05
Neoprene Rubber
126.407 ± 198.446
300.259 ±82.964
f0.00± 331.89
„ 30.31 ±
72.96 ±
1.16 14DayS
Optical Plastic
320
193.05 ± 133.936
201.572 ± 148.937
f0.00 ± 92.76
8 0.21
Bare Pine Wood
13.848 ±4.799
18.291 ± 8.236
f0.00 ± 65.79
Industrial Carpet
85.453 ±35.247
49.905 ± 34.604
41.60 ±41.30
Paper
31.675 ± 12.263
7.440 ±0.312
76.51 ±8.02
Mild Steel
151.614 ± 36.500
241.433 ±47.482
f0.00 ± 43.39
Neoprene Rubber
448.312 ± 162.928
481.273 ± 106.865
f0.00 ± 40.08
20.41 ±
45.22 ±
j 47 7 Days
Optical Plastic
320
311.765 ±249.232
359.029 ±23.014
f0.00 ± 80.95
9 0.23
Bare Pine Wood
31.458 ±8.052
88.154 ±27.781
f0.00 ± 99.72
Industrial Carpet
343.024 ± 109.283
392.103 ± 147.069
f0.00± 49.31
Paper
337.619 ± 162.887
376.756 ±65.259
f0.00± 50.14
Mild Steel
151.614 ± 36.500
92.125 ±29.819
39.24 ±21.48
Neoprene Rubber
448.312 ± 162.928
497.513 ± 147.321
f0.00 ± 45.60
10
20.59 ±
0.29
45.26 ±
1.40
14 Days
Optical Plastic
Bare Pine Wood
Industrial Carpet
Paper
320
311.765 ±249.232
31.458 ±8.052
343.024 ± 109.283
337.619 ± 162.887
474.946 ±96.155
52.176 ± 18.768
407.41 ±51.057
333.034 ±34.917
f0.00± 110.12
f0.00± 64.18
f0.00± 35.64
1.36 ±42.69
Mild Steel
507.441 ±61.225
58.110 ± 15.498
88.55 ±2.94
Neoprene Rubber
651.201 ± 123.619
461.810 ±206.330
29.08 ±30.18
11
20.80 ±
75.28 ±
7 Days
Optical Plastic
320
488.828 ±280.165
352.736 ± 53.685
27.84 ±37.51
0.55
1.05
Bare Pine Wood
Industrial Carpet
Paper
124.416 ±28.624
843.428 ±221.927
533.712 ±34.554
46.347 ± 10.805
585.063 ±306.596
280.187 ±97.739
62.75 ± 10.69
30.63 ±35.65
47.50 ± 16.33
Mild Steel
507.441 ±61.225
27.470 ± 8.556
94.59 ± 1.58
Neoprene Rubber
651.201 ± 123.619
364.574 ±79.840
44.02 ± 14.22
12
20.84 ±
0.80
72.43 ±
5.14
14 Days
Optical Plastic
Bare Pine Wood
Industrial Carpet
Paper
320
488.828 ±280.165
124.416 ±28.624
843.428 ±221.927
533.712 ±34.554
429.044 ± 17.369
37.655 ± 12.467
428.759 ±43.394
327.182 ±68.640
12.23 ± 44.20
69.73 ± 10.70
49.16 ± 12.56
38.70 ± 11.80
Mild Steel
327.676 ±81.598
127.425 ±60.295
61.11 ± 18.23
Neoprene Rubber
453.217 ± 55.596
417.753 ±93.573
7.82 ±20.63
13
19.80 ±
0.53
44.81 ±
4.03
21 Days
Optical Plastic
Bare Pine Wood
Industrial Carpet
Paper
320
427.380 ±21.900
95.760 ± 13.843
655.811 ± 167.921
421.922 ±51.930
423.727 ±46.716
37.268 ± 11.525
36.078 ± 20.466
139.322 ±50.402
0.85 ± 10.57
61.08 ± 11.65
94.50 ±3.00
66.98 ± 11.06
3 Data are expressed as the mean (± SD) of the mass of toxin recovered on five replicate individual samples, and attenuation (percent reduction ± CI).
b Positive Controls = samples inoculated, not attenuated.
c Test Coupons = samples inoculated, attenuated.
d CI = confidence interval (± 1.96 x standard error [SE]).
As a result of high variability, negative value reported as "0".
A-5
-------
Table A-2. Attenuation of Crude Ricin Toxin" (Continued)
lost
X Limber
Tosl Parameters
Temp :C
Material
%RII
lime
Inociilimi
(ug coupon)
Mean Recovered Ricin ± SD
(|ig coupon)
Positive Control
Test Coupon
"^Reduction
CI
Mild Steel
327.676 81.598
124.88 39.143
61.89 13.37
Neoprene Rubber
453.217 + 55.596
141.939 + 92.140
68.68+ 18.14
14
19.82 +
0.47
45.06 +
4.18
28 Days
Optical Plastic
Bare Pine Wood
Industrial Carpet
Paper
320
427.380 + 21.900
95.760+ 13.843
655.811 + 167.921
421.922 + 51.930
36.307 + 29.923
27.814+ 10.679
157.749 + 88.907
75.495 + 35.585
91.50 + 6.15
70.95 + 10.45
75.95 + 13.05
82.11 + 7.64
Mild Steel TO
345.189 + 30.327
NA
NA
Mild Steel +6 h
213.405 + 33.609
38.18 + 9.77
15
50.26 +
0.24
21.05 +
2.67
Multiple
Mild Steel +24 h
Mild Steel +30 h
Mild Steel +48 h
Mild Steel +72 h
Mild Steel +96 h
320
NAg
124.504 + 56.319
258.024 + 63.255
213.280 + 35.543
185.231 + 53.836
210.139 + 92.689
63.93+ 14.57
25.25+ 17.06
38.21 + 10.20
46.34+ 14.28
39.12 + 24.00
Mild Steel TO
326.962 + 35.215
NA
NA
Mild Steel +48 h
256.930 + 62.722
21.42+ 18.38
16
39.95 +
0.43
26.62
+3.31
Multiple
Mild Steel +72 h
Mild Steel +96 h
Mild Steel +120 h
Mild Steel +144 h
Mild Steel +168 h
320
NA
308.604 + 48.759
331.161 + 36.136
295.797 + 40.980
196.584 + 27.157
244.837 + 34.795
5.61 + 15.82
0.00+13.61
9.53 + 13.92
39.88 + 9.23
25.12+ 11.70
Mild Steel TO
358.674 + 48.186
NA
NA
Mild Steel +48 h
163.327 + 82.796
54.42 + 20.93
17
50.41 +
0.72
19.79 +
2.20
Multiple
Mild Steel +72 h
Mild Steel +96 h
Mild Steel +120 h
Mild Steel+144 h
Mild Steel +168 h
320
NA
184.327 + 27.940
145.681 + 149.787
170.249 + 97.208
171.211 + 37.702
6.661 + 3.393
48.61 + 9.12
59.38 + 36.92
52.53 + 24.40
52.27+ 10.79
98.14 + 0.86
18
40.37 ±
0.49
21.56 ±
2.48
Mild Steel TO
Mild Steel +3 Days
Mild Steel +4 Days
Mild Steel +5 Days
Mild Steel +6 Days
Multiple Mild Steel +7 Days
Mild Steel +10 Days
Mild Steel +11 Days
Mild Steel +12 Days
Mild Steel +13 Days
Mild Steel +14 Days
200.575 ± 44.939
320
NA
NA
279.570+ 105.000
216.049 + 97.292
321.147 + 381.795
48.251 + 17.368
104.119+ 103.739
229.036+ 135.947
49.024 + 58.435
94.181 + 100.927
41.227 + 60.082
104.536 + 31.373
NA
0.00 + 53.43
0.00 + 47.49
0.00+ 169.79
75.94 + 8.94
48.09 + 46.47
0.00 + 63.50
75.56 + 25.98
53.04 + 45.06
79.45 + 26.57
47.88+ 17.11
a Data are expressed as the mean (+ SD) of the mass of toxin observed on five individual samples, and attenuation (percent reduction ± CI).
b Positive Controls = samples inoculated, not attenuated.
c Test Coupons = samples inoculated, attenuated.
11 CI = confidence interval (+ 1.96 x standard error [SE]).
f As a result of high variability, negative value reported as "0".
gNA = Not applicable.
A-6
-------
Appendix B
Detailed Statistical Analysis
Introduction
This report contains the statistical analysis of ricin percent reduction data over time
generated from pure and crude ricin preparations on mild steel, rubber, plastic, wood, carpet, and
paper at various temperatures and percent humidity.
Results
The ANOVA models fitted with effects for material, ricin type and time were fitted to
each combination of temperature and relative humidity. These models were used to generate
estimates and 95 percent confidence intervals for the percent reduction at each combination of
material, ricin type and time. Tables B-la through B-li present these results and a p-value
testing whether the percent reduction was significantly different from zero. Figures B-la
through B-li plot the descriptive statistics and the individual percent reductions by material, ricin
type and time point for each combination of temperature and relative humidity. Figures B-2a
through B-2f plot the descriptive statistics and the individual percent reductions by material, ricin
type and relative humidity for each combination of temperature and time.
The same ANOVA models were used to test for significant differences between different
combinations of material, ricin type, and time. The tables of results comparing the percent
reductions for varying conditions show unadjusted and Tukey's adjusted p-values for each
comparison of interest. An up or down arrow is included indicating whether the specific level of
the factor had a percent reduction greater or less than that for the level being compared. Given
the large number of comparisons, it is recommended that the Tukey's adjusted p-values be used
for interpreting the results. If the unadjusted p-values are used, it is likely that there would be a
large number of significant comparisons from random variability when no true difference
between the conditions exists.
Tables B-2a through B-2g present results from the models testing for significant
differences among the materials for each combination of temperature, humidity, time, and ricin
type. Tables B-3a through B-3g present results from the models testing for significant
differences among the time points for each combination of temperature, humidity, material, and
ricin type. Tables B-4a through B-4h present results from the models testing for significant
differences among the ricin types for each combination of temperature, humidity, material, and
time point.
For each ricin type and material, separate ANOVA models were fitted with effects for
temperature, relative humidity, and time. Tables B-5a and B-5b present results from this second
set of models testing for significant differences among the different levels of humidity for each
combination of temperature and time. For each ricin type and material, Tables B-6a through B-
B-l
-------
6g present results from this second set of models testing for significant differences among the
different temperatures for each combination of relative humidity and time.
B-l
-------
Table B-1a. Mean Percent Reduction, 95% Confidence Interval, and P-value for Crude
Ricin, by Material and Time at 20 °C and 45% Humidity.
Temperature
(°C)
Humidity
(%)
Ricin
Type
Material
Time
Percent Reduction
Estimate and 95%
Confidence Interval
P-Value
20
45
Crude
Carpet
7 Days
-8.12 (-82.82, 36.06)
0.7697
14 Days
-18.07 (-99.65, 30.17)
0.5335
21 Days
95.39 (92.20, 97.27)
<0.0001*
28 Days
79.22 (64.87, 87.71)
<0.0001*
Paper
7 Days
-10.37 (-86.62, 34.73)
0.7115
14 Days
1.78 (-66.08, 41.92)
0.9462
21 Days
68.77 (47.19, 81.53)
<0.0001*
28 Days
83.57 (72.22, 90.29)
<0.0001*
Plastic
7 Days
-10.47 (-86.80, 34.67)
0.7089
14 Days
-50.24 (-154.05, 11.15)
0.1280
21 Days
8.11 (-55.38, 45.66)
0.7512
28 Days
89.81 (82.77, 93.97)
<0.0001*
Rubber
7 Days
-5.33 (-78.11, 37.71)
0.8455
14 Days
21 Days
-7.26 (-81.37, 36.57)
10.08 (-52.04, 46.82)
0.7926
0.6902
28 Days
73.59 (55.34, 84.38)
<0.0001*
Steel
Wood
7 Days
14 Days
-56.38 (-164.42, 7.52)
41.62 (1.29, 65.48)
0.0948
0.0447*
21 Days
64.78 (40.45, 79.17)
0.0001*
28 Days
7 Days
63.59 (38.44, 78.47)
-169.02 (-354.90, -59.10)
0.0002*
0.0003*
14 Days
-56.17 (-164.07, 7.64)
0.0958
21 Days
28 Days
62.28 (36.22, 77.69)
72.50 (53.50, 83.74)
0.0003*
<0.0001*
'Significant at the 0.05 level.
B-l
-------
Table B-1b. Mean Percent Reduction, 95% Confidence Interval, and P-value for Pure
Ricin, by Material and Time at 20 °C and 45% Humidity.
Temperature
(°C)
Humidity
(%)
Ricin
Type
Material
Time
Percent Reduction
Estimate and 95%
Confidence Interval
P-Value
20
45
Pure
Carpet
7 Days
47.37 (11.00, 68.87)
0.0169*
14 Days
21 Days
93.84 (89.59, 96.36)
74.67 (57.17, 85.02)
<0.0001*
<0.0001*
28 Days
90.74 (84.35, 94.52)
<0.0001*
Paper
7 Days
46.82 (10.08, 68.55)
0.0187*
14 Days
44.91 (6.84, 67.42)
0.0263*
21 Days
50.57 (16.42, 70.77)
0.0088*
28 Days
68.28 (46.36, 81.24)
<0.0001*
Plastic
7 Days
92.48 (87.29, 95.55)
<0.0001*
14 Days
78.63 (63.86, 87.36)
<0.0001*
21 Days
87.19(78.34, 92.42)
<0.0001*
28 Days
91.83 (86.18, 95.17)
<0.0001*
Rubber
7 Days
14 Days
12.46 (-48.02, 48.23)
62.54 (36.66, 77.85)
0.6178
0.0003*
21 Days
86.33 (76.89, 91.92)
<0.0001*
28 Days
89.28 (81.88, 93.66)
<0.0001*
Steel
7 Days
75.77 (59.03, 85.67)
<0.0001*
14 Days
97.08 (95.07, 98.28)
<0.0001*
21 Days
92.75 (87.74, 95.71)
<0.0001*
28 Days
99.00 (98.30, 99.41)
<0.0001*
Wood
7 Days
-25.36 (-111.98, 25.86)
0.3971
14 Days
-44.21 (-143.85, 14.71)
0.1708
21 Days
68.12 (46.09, 81.15)
<0.0001*
28 Days
42.91 (3.46, 66.24)
0.0366*
'Significant at the 0.05 level.
B-l
-------
Table B-1c. Mean Percent Reduction, 95% Confidence Interval, and P-value by Ricin
Type, Material, and Time at 20 °C and 75% Humidity.
Temperature
(°C)
Humidity
(%)
Ricin
Type
Material
Time
Percent Reduction
Estimate and 95%
Confidence Interval
P-Value
20
75
Crude
Carpet
7 Days
37.61 (12.43, 55.55)
0.0069*
14 Days
49.38 (28.94, 63.94)
0.0001*
Paper
Plastic
7 Days
50.04 (29.87, 64.41)
<0.0001*
14 Days
7 Days
39.77 (15.45, 57.09)
48.16 (27.24, 63.07)
0.0038*
0.0002*
14 Days
34.11 (7.51, 53.06)
0.0164*
Rubber
7 Days
33.51 (6.67, 52.63)
0.0188*
14 Days
45.01 (22.81, 60.82)
0.0007*
Steel
7 Days
88.87 (84.37, 92.07)
<0.0001*
14 Days
94.76 (92.64, 96.27)
<0.0001*
Wood
7 Days
63.46 (48.71, 73.97)
<0.0001*
14 Days
71.09 (59.42, 79.41)
<0.0001*
Pure
Carpet
7 Days
29.85(1.53, 50.02)
0.0407*
14 Days
64.17 (49.71, 74.47)
<0.0001*
Paper
7 Days
14 Days
5.38 (-32.81, 32.59)
57.31 (40.07, 69.58)
0.7466
<0.0001*
Plastic
7 Days
-12.54 (-57.97, 19.82)
0.4907
14 Days
65.45 (51.50, 75.38)
<0.0001*
Rubber
7 Days
12.91 (-22.25, 37.95)
0.4205
14 Days
65.26 (51.23, 75.25)
<0.0001*
Steel
7 Days
97.78 (96.89, 98.42)
<0.0001*
14 Days
98.58 (98.01, 98.99)
<0.0001*
Wood
7 Days
-305.49 (-469.17, -188.88)
<0.0001*
14 Days
-50.87 (-111.77, -7.48)
0.0180*
'Significant at the 0.05 level.
B-l
-------
Table B-1d. Mean Percent Reduction, 95% Confidence Interval, and P-value by Ricin
Type, Material, and Time at 25 °C and 45% Humidity.
Temperature
(°C)
Humidity
(%)
Ricin
Type
Material
Time
Percent Reduction
Estimate and 95%
Confidence Interval
P-Value
25
45
Crude
Carpet
7 Days
-28.16 (-131.11, 28.93)
0.4056
14 Days
81.05 (65.82, 89.49)
<0.0001*
Paper
Plastic
7 Days
93.84 (88.88, 96.58)
<0.0001*
14 Days
7 Days
95.15 (91.25, 97.31)
52.01 (13.46, 73.39)
<0.0001*
0.0152*
14 Days
72.19 (49.85, 84.58)
<0.0001*
Rubber
7 Days
54.53 (18.00, 74.78)
0.0093*
14 Days
88.61 (79.46, 93.68)
<0.0001*
Steel
7 Days
40.22 (-7.81, 66.85)
0.0865
14 Days
38.66 (-10.61, 65.99)
0.1031
Wood
7 Days
47.72 (5.72, 71.01)
0.0315*
14 Days
60.54 (28.84, 78.12)
0.0023*
Pure
Carpet
7 Days
43.85 (-1.26, 68.86)
0.0550
14 Days
95.43 (91.75, 97.46)
<0.0001*
Paper
7 Days
14 Days
61.19 (30.02, 78.48)
95.40 (91.71, 97.45)
0.0019*
<0.0001*
Plastic
7 Days
82.89 (69.15, 90.51)
<0.0001*
14 Days
95.87 (92.56, 97.71)
<0.0001*
Rubber
7 Days
17.61 (-48.58, 54.31)
0.5159
14 Days
82.86 (69.09, 90.49)
<0.0001*
Steel
7 Days
88.68 (79.58, 93.72)
<0.0001*
14 Days
96.74 (94.12, 98.19)
<0.0001*
Wood
7 Days
-35.80 (-144.90, 24.69)
0.3055
14 Days
64.19 (35.43, 80.14)
0.0008*
'Significant at the 0.05 level.
B-l
-------
Table B-1e. Mean Percent Reduction, 95% Confidence Interval, and P-value by Ricin
Type, Material, and Time at 25 °C and 75% Humidity.
Temperature
(°C)
Humidity
(%)
Ricin
Type
Material
Time
Percent Reduction
Estimate and 95%
Confidence Interval
P-Value
25
75
Crude
Carpet
7 Days
94.16 (88.38, 97.06)
<0.0001*
14 Days
97.71 (95.44, 98.85)
<0.0001*
Paper
Plastic
7 Days
99.54 (99.08, 99.77)
<0.0001*
14 Days
7 Days
96.59 (93.22, 98.29)
84.44 (69.05, 92.18)
<0.0001*
<0.0001*
14 Days
-243.63 (-583.68, -72.71)
0.0006*
Rubber
7 Days
12.65 (-73.79, 56.10)
0.6972
14 Days
12.89 (-73.31, 56.22)
0.6914
Steel
7 Days
83.52 (67.21, 91.72)
<0.0001*
14 Days
96.19 (92.43, 98.09)
<0.0001*
Wood
7 Days
49.83 (0.18, 74.78)
0.0494*
14 Days
-25.49 (-149.68, 36.92)
0.5139
Pure
Carpet
7 Days
73.49 (47.26, 86.68)
0.0002*
14 Days
94.06 (88.17, 97.01)
<0.0001*
Paper
7 Days
14 Days
67.31 (34.97, 83.57)
95.03 (90.12, 97.50)
0.0017*
<0.0001*
Plastic
7 Days
24.98 (-49.25, 62.30)
0.4089
14 Days
58.55 (17.53, 79.17)
0.0127*
Rubber
7 Days
77.75 (55.74, 88.82)
<0.0001*
14 Days
93.35 (86.76, 96.66)
<0.0001*
Steel
7 Days
99.88 (99.77, 99.94)
<0.0001*
14 Days
99.95 (99.91, 99.98)
<0.0001*
Wood
7 Days
70.33 (40.97, 85.09)
0.0007*
14 Days
89.41 (78.94, 94.68)
<0.0001*
'Significant at the 0.05 level.
B-l
-------
Table B-1f. Mean Percent Reduction, 95% Confidence Interval, and P-value by Ricin
Type, Material, and Time at 30 °C and 45% Humidity.
Temperature
(°C)
Humidity
(%)
Ricin
Type
Material
Time
Percent Reduction
Estimate and 95%
Confidence Interval
P-Value
30
45
Crude
Carpet
7 Days
92.93 (82.05, 97.21)
<0.0001*
14 Days
94.34 (85.63, 97.77)
<0.0001*
Paper
Plastic
7 Days
-134.36 (-494.83, 7.67)
0.0726
14 Days
7 Days
64.05 (8.76, 85.84)
95.90 (89.60, 98.39)
0.0317*
<0.0001*
14 Days
88.92 (71.88, 95.63)
<0.0001*
Rubber
7 Days
92.13 (80.02, 96.90)
<0.0001*
14 Days
82.57 (55.77, 93.13)
0.0003*
Steel
7 Days
59.75 (-2.17, 84.14)
0.0554
14 Days
87.64 (68.63, 95.13)
<0.0001*
Wood
7 Days
36.58 (-60.98, 75.01)
0.3343
14 Days
69.55 (22.72, 88.00)
0.0129*
Pure
Carpet
7 Days
99.50 (98.73, 99.80)
<0.0001*
14 Days
61.38(1.99, 84.79)
0.0454*
Paper
7 Days
14 Days
99.93 (99.82, 99.97)
93.29 (82.96, 97.36)
<0.0001*
<0.0001*
Plastic
7 Days
78.80 (46.19, 91.65)
0.0013*
14 Days
92.02 (79.74, 96.86)
<0.0001*
Rubber
7 Days
92.00 (79.68, 96.85)
<0.0001*
14 Days
81.79 (53.79, 92.83)
0.0005*
Steel
7 Days
84.37 (60.34, 93.84)
0.0001*
14 Days
92.10 (79.96, 96.89)
<0.0001*
Wood
7 Days
89.08 (72.29, 95.70)
<0.0001*
14 Days
30.54 (-76.29, 72.64)
0.4392
'Significant at the 0.05 level.
B-l
-------
Table B-1g. Mean Percent Reduction, 95% Confidence Interval, and P-value by Ricin
Type, Material, and Time at 30 °C and 75% Humidity.
Temperature
(°C)
Humidity
(%)
Ricin
Type
Material
Time
Percent Reduction
Estimate and 95%
Confidence Interval
P-Value
30
75
Crude
Carpet
7 Days
3.71 (-103.85, 54.52)
0.9205
14 Days
54.46 (3.59, 78.49)
0.0400*
Paper
Plastic
7 Days
94.33 (88.00, 97.32)
<0.0001*
14 Days
7 Days
76.53 (50.31, 88.91)
56.45 (7.80, 79.43)
0.0002*
0.0302*
14 Days
8.61 (-93.49, 56.83)
0.8122
Rubber
7 Days
31.40 (-45.23, 67.60)
0.3211
14 Days
-131.30 (-389.70, -9.25)
0.0288*
Steel
7 Days
98.42 (96.66, 99.26)
<0.0001*
14 Days
94.59 (88.54, 97.44)
<0.0001*
Wood
7 Days
54.57 (3.83, 78.54)
0.0394*
14 Days
-21.79 (-157.84, 42.47)
0.6031
Pure
Carpet
7 Days
82.29 (62.50, 91.63)
<0.0001*
14 Days
72.57 (41.93, 87.04)
0.0009*
Paper
7 Days
14 Days
94.81 (89.01, 97.55)
75.62 (48.38, 88.48)
<0.0001*
0.0003*
Plastic
7 Days
95.77 (91.05, 98.00)
<0.0001*
14 Days
71.48 (39.62, 86.53)
0.0013*
Rubber
7 Days
75.66 (48.46, 88.50)
0.0003*
14 Days
60.78 (16.97, 81.48)
0.0150*
Steel
7 Days
94.78 (88.96, 97.54)
<0.0001*
14 Days
99.90 (99.80, 99.95)
<0.0001*
Wood
7 Days
70.08 (36.67, 85.87)
0.0019*
14 Days
-232.49 (-603.92, -57.05)
0.0020*
'Significant at the 0.05 level.
B-l
-------
Table B-1h. Mean Percent Reduction, 95% Confidence Interval, and P-value for Steel, by
Ricin Type and Time at 40 °C and 20% Humidity.
Temperature
(°C)
Humidity
(%)
Ricin
Type
Material
Time
Percent Reduction
Estimate and 95%
Confidence Interval
P-Value
40
20
Crude
Steel
48 Hours
23.16 (-74.05, 66.08)
0.5239
72 Hours
-10.32 (-96.67, 38.12)
0.7366
96 Hours
0.90 (-76.68, 44.41)
0.9754
120 Hours
144 Hours
14.51 (-52.41, 52.04)
63.66 (35.22, 79.62)
0.5916
0.0008*
7 Days
55.37 (20.44, 74.97)
0.0067*
10 Days
41.54 (-32.42, 74.19)
0.1955
11 Days
83.82 (63.36, 92.86)
<0.0001*
12 Days
79.21 (52.92, 90.82)
0.0002*
13 Days
91.70 (81.21, 96.34)
<0.0001*
14 Days
49.96 (-13.35, 77.91)
0.0960
Pure
Steel
48 Hours
90.51 (78.51, 95.81)
<0.0001*
72 Hours
93.51 (85.30, 97.14)
<0.0001*
96 Hours
94.80 (88.22, 97.70)
<0.0001*
120 Hours
144 Hours
96.86 (92.89, 98.61)
97.00 (93.20, 98.68)
<0.0001*
<0.0001*
7 Days
96.87 (92.90, 98.62)
<0.0001*
'Significant at the 0.05 level.
B-l
-------
Table B-1i. Mean Percent Reduction, 95% Confidence Interval, and P-value for Steel, by
Ricin Type and Time at 50 °C and 20% Humidity.
Temperature
(°C)
Humidity
(%)
Ricin
Type
Material
Time
Percent Reduction
Estimate and 95%
Confidence Interval
P-Value
50
20
Crude
Steel
6 Hours
38.74 (3.43, 61.14)
0.0351*
24 Hours
67.94 (49.47, 79.66)
<0.0001*
30 Hours
31.53 (-7.93, 56.57)
0.1018
48 Hours
72 Hours
50.99 (32.38, 64.48)
48.63 (29.13, 62.77)
<0.0001*
<0.0001*
96 Hours
64.69 (51.28, 74.40)
<0.0001*
120 Hours
60.45 (37.66, 74.91)
0.0001*
144 Hours
53.24 (26.29, 70.34)
0.0013*
7 Days
98.29 (97.30, 98.91)
<0.0001*
Pure
Steel
6 Hours
63.36 (42.24, 76.75)
<0.0001*
24 Hours
86.01 (77.95, 91.13)
<0.0001*
30 Hours
90.96 (85.76, 94.27)
<0.0001*
48 Hours
95.55 (93.85, 96.77)
<0.0001*
72 Hours
98.10(97.38, 98.62)
<0.0001*
96 Hours
120 Hours
98.60 (98.07, 98.99)
99.12 (98.62, 99.44)
<0.0001*
<0.0001*
144 Hours
99.17 (98.69, 99.47)
<0.0001*
7 Days
99.93 (99.88, 99.95)
<0.0001*
'Significant at the 0.05 level.
B-l
-------
Table B-2a. Unadjusted and Tukey Adjusted P-values Comparing Materials for 20 °C and 45% Humidity, Crude Ricin by
Time.
Unadjusted P-Value
Tukey Adjusted P-Value
Ricin
Type
Temperature
(°C)
Humidity
(%)
Time
Material
Carpet
Paper
Plastic
Rubber
Steel
Carpet
Paper
Plastic
Rubber
Steel
Paper
0.9565
1.0000
Plastic
0.9545
0.9980
1.0000
1.0000
7 Days
Rubber
0.9448
0.9015
0.8995
1.0000
1.0000
1.0000
Steel
0.3284
0.3560
0.3573
0.2954
1.0000
1.0000
1.0000
1.0000
Wood
0.0164* |
0.0190* |
0.0191* |
0.0136* |
0.1514
0.9525
0.9654
0.9659
0.9316
1.0000
Paper
0.6255
1.0000
Plastic
0.5231
0.2605
1.0000
1.0000
14 Days
Rubber
0.7991
0.8153
0.3721
1.0000
1.0000
1.0000
Steel
0.0630
0.1688
0.0129* t
0.1079
0.9995
1.0000
0.9243
1.0000
Crude
20
45
Wood
0.4587
0.2197
0.9183
0.3198
0.0097* |
1.0000
1.0000
1.0000
1.0000
0.8800
Paper
<0.0001* |
0.0009* |
Plastic
<0.0001* |
0.0046* |
<0.0001* |
0.7179
21 Days
Rubber
<0.0001* |
0.0055* |
0.9540
<0.0001* |
0.7607
1.0000
Steel
<0.0001* |
0.7502
0.0117* t
0.0137* t
0.0002* |
1.0000
0.9100
0.9318
Wood
<0.0001* |
0.6169
0.0191* f
0.0221* t
0.8556
<0.0001* |
1.0000
0.9657
0.9762
1.0000
Paper
0.5335
1.0000
Plastic
0.0600
0.2062
0.9994
1.0000
28 Days
Rubber
0.5249
0.2089
0.0122* |
1.0000
1.0000
0.9171
Steel
0.1380
0.0359* |
0.0009* |
0.3950
1.0000
0.9945
0.3104
1.0000
Wood
0.4575
0.1728
0.0091* |
0.9145
0.4572
1.0000
1.0000
0.8679
1.0000
1.0000
* Significant at the 0.05 level.
T, | "T" indicates the mean percent reduction of the row material is significantly greater than the column material, while "J," indicates the mean percent
reduction of the row material is significantly less than the column material.
B-13
-------
Table B-2b. Unadjusted and Tukey Adjusted P-values Comparing Materials for 20 °C and 45% Humidity, Pure Ricin by
Time.
Unadjusted P-Value
Tukey Adjusted P-Value
Ricin
Type
Temperature
(°C)
Humidity
(%)
Time
Material
Carpet
Paper
Plastic
Rubber
Steel
Carpet
Paper
Plastic
Rubber
Steel
Pure
20
45
7 Days
Paper
0.9782
1.0000
Plastic
<0.0001* f
<0.0001* t
0.0006* f
0.0005* t
Rubber
0.1784
0.1873
<0.0001* 1
1.0000
1.0000
<0.0001* 1
Steel
0.0408* f
0.0382* t
0.0022* |
0.0008* f
0.9966
0.9956
0.5201
0.2914
Wood
0.0223* |
0.0239* |
<0.0001* |
0.3415
<0.0001* 1
0.9765
0.9805
<0.0001* |
1.0000
0.0161* |
14
Days
Paper
<0.0001* |
<0.0001* |
Plastic
0.0011* |
0.0127* t
0.3649
0.9227
Rubber
Steel
<0.0001* |
0.0486* f
0.3070
<0.0001* f
0.1378
<0.0001* f
<0.0001* f
0.0029* |
0.9984
1.0000
<0.0001* f
1.0000
0.0003* t
<0.0001* f
Wood
<0.0001* |
0.0114* |
<0.0001* |
0.0004* |
<0.0001* 1
<0.0001* |
0.9065
0.0009* |
0.1945
<0.0001* |
21
Days
Paper
0.0774
0.9999
Plastic
0.0718
0.0004* f
0.9998
0.1915
Rubber
0.1030
0.0008* f
0.8637
1.0000
0.2893
1.0000
Steel
0.0011* f
<0.0001* f
0.1325
0.0941
0.3528
0.0008* f
1.0000
1.0000
Wood
0.5420
0.2457
0.0164* |
0.0256* |
0.0001* 1
1.0000
1.0000
0.9523
0.9839
0.0712
28
Days
Paper
Plastic
0.0013* |
0.7409
0.0004* f
0.3906
1.0000
0.1840
Rubber
0.6984
0.0044* f
0.4729
1.0000
0.7054
1.0000
Steel
<0.0001* f
<0.0001* f
<0.0001* f
<0.0001* f
<0.0001* f
<0.0001* f
<0.0001* f
<0.0001* f
Wood
<0.0001* |
0.1203
<0.0001* |
<0.0001* |
<0.0001* 1
0.0025* |
1.0000
0.0006* |
0.0120* |
<0.0001* |
* Significant at the 0.05 level.
T, | "T" indicates the mean percent reduction of the row material is significantly greater than the column material, while "J," indicates the mean percent
reduction of the row material is significantly less than the column material.
B-14
-------
Table B-2c. Unadjusted and Tukey Adjusted P-values Comparing Materials for 20 °C and 75% Humidity, Crude and Pure
Ricin by Time.
Unadjusted P-Value
Tukey Adjusted P-Value
Ricin
Type
Temperature
(°C)
Humidity
(%)
Time
Material
Carpet
Paper
Plastic
Rubber
Steel
Carpet
Paper
Plastic
Rubber
Steel
Crude
20
75
7
Days
Paper
0.3602
1.0000
Plastic
0.4452
0.8789
1.0000
1.0000
Rubber
0.7926
0.2397
0.3055
1.0000
0.9999
1.0000
Steel
<0.0001* f
<0.0001* f
<0.0001* f
<0.0001* t
<0.0001* t
<0.0001* f
<0.0001* f
<0.0001* f
Wood
0.0292* t
0.1986
0.1510
0.0150* t
<0.0001* 1
0.8530
0.9998
0.9988
0.6890
0.0008* |
14
Days
Paper
0.4735
1.0000
Plastic
0.2780
0.7111
1.0000
1.0000
Rubber
0.7325
0.7071
0.4561
1.0000
1.0000
1.0000
Steel
<0.0001* f
<0.0001* f
<0.0001* f
<0.0001* f
<0.0001* t
<0.0001* f
<0.0001* f
<0.0001* f
Wood
0.0225* t
0.0031* t
0.0010* f
0.0091* f
<0.0001* 1
0.7943
0.2928
0.1256
0.5526
<0.0001* |
Pure
20
75
7
Days
Paper
0.2187
0.9999
Plastic
0.0533
0.4743
0.9501
1.0000
Rubber
0.3729
0.7324
0.2912
1.0000
1.0000
1.0000
Steel
<0.0001* f
<0.0001* f
<0.0001* f
<0.0001* f
<0.0001* f
<0.0001* f
<0.0001* f
<0.0001* f
Wood
<0.0001* |
<0.0001* |
<0.0001* |
<0.0001* 1
<0.0001* 1
<0.0001* |
<0.0001* |
0.0002* |
<0.0001* |
<0.0001* |
14
Days
Paper
Plastic
0.4699
0.8810
0.3834
1.0000
1.0000
1.0000
Rubber
0.8989
0.3958
0.9820
1.0000
1.0000
1.0000
Steel
<0.0001* f
<0.0001* f
<0.0001* f
<0.0001* f
<0.0001* f
<0.0001* t
<0.0001* f
<0.0001* f
Wood
<0.0001* |
<0.0001* |
<0.0001* |
<0.0001* 1
<0.0001* 1
<0.0001* 1
0.0002* 1
<0.0001* 1
<0.0001* |
<0.0001* 1
* Significant at the 0.05 level.
T, | "T" indicates the mean percent reduction of the row material is significantly greater than the column material, while "J," indicates the mean percent
reduction of the row material is significantly less than the column material.
B-15
-------
Table B-2d. Unadjusted and Tukey Adjusted P-values Comparing Materials for 25 °C and45% Humidity, Crude and Pure
Ricin by Time.
Unadjusted P-Value
Tukey Adjusted P-Value
Ricin
Type
Temperature
(°C)
Humidity
(%)
Time
Material
Carpet
Paper
Plastic
Rubber
Steel
Carpet
Paper
Plastic
Rubber
Steel
Crude
25
45
7
Days
Paper
<0.0001* t
<0.0001*
T
Plastic
0.0215* t
<0.0001* 1
0.7826
0.0010* 1
Rubber
0.0154* t
<0.0001* 1
0.8982
0.6969
0.0016* |
1.0000
Steel
0.0726
<0.0001* 1
0.6022
0.5164
0.9772
0.0001* |
1.0000
1.0000
Wood
0.0354* t
<0.0001* 1
0.8389
0.7405
0.7503
0.8903
0.0004* |
1.0000
1.0000
1.0000
14
Days
Paper
0.0016* t
0.1880
Plastic
0.3638
<0.0001* 1
1.0000
0.0136* |
Rubber
0.2283
0.0449* |
0.0362* t
0.9999
0.9286
0.8943
Steel
0.0063* |
<0.0001* |
0.0627
0.0001* |
0.4535
<0.0001*
0.9662
0.0222* |
Wood
0.0841
<0.0001* 1
0.4068
0.0039* |
0.2964
0.9854
0.0006* |
1.0000
0.3422
1.0000
Pure
25
45
7
Days
Paper
0.3813
1.0000
Plastic
0.0057* t
0.0541
0.4294
0.9518
Rubber
0.3637
0.0762
0.0003* |
1.0000
0.9802
0.0505
Steel
0.0002* t
0.0042* t
0.3282
<0.0001* f
0.0410* f
0.3589
1.0000
0.0018* f
Wood
0.0381* |
0.0036* |
<0.0001* |
0.2371
<0.0001* |
0.9034
0.3272
0.0008* |
0.9999
<0.0001* 1
14
Days
Paper
Plastic
0.9901
0.8074
0.7978
1.0000
1.0000
1.0000
Rubber
0.0022* |
0.0023* |
0.0010* |
0.2348
0.2412
0.1325
Steel
0.4230
0.4159
0.5767
0.0001* f
1.0000
1.0000
1.0000
0.0268* t
Wood
<0.0001* |
<0.0001* |
<0.0001* |
0.0827
<0.0001* |
0.0009* |
0.0010* |
0.0003* |
0.9846
<0.0001* 1
* Significant at the 0.05 level.
T, | "T" indicates the mean percent reduction of the row material is significantly greater than the column material, while "J," indicates the mean percent
reduction of the row material is significantly less than the column material.
B-16
-------
Table B-2e. Unadjusted and Tukey Adjusted P-values Comparing Materials for 25 °C and 75% Humidity, Crude and Pure
Ricin by Time.
Unadjusted P-Value
Tukey Adjusted P-Value
Ricin
Type
Temperature
(°C)
Humidity
(%)
Time
Material
Carpet
Paper
Plastic
Rubber
Steel
Carpet
Paper
Plastic
Rubber
Steel
Crude
25
75
7
Days
Paper
<0.0001* t
0.0003* t
Plastic
0.0485* |
<0.0001* 1
0.9389
<0.0001* 1
Rubber
<0.0001* |
<0.0001* 1
0.0007* |
<0.0001* |
<0.0001* 1
0.0943
Steel
0.0369* |
<0.0001* 1
0.9066
0.0010* f
0.8980
<0.0001* 1
1.0000
0.1281
Wood
<0.0001* |
<0.0001* 1
0.0188* |
0.2608
0.0254* |
0.0062* |
<0.0001* 1
0.7500
1.0000
0.8223
14
Days
Paper
Plastic
0.4216
<0.0001* |
<0.0001* 1
1.0000
<0.0001* |
<0.0001* 1
Rubber
<0.0001* |
<0.0001* 1
0.0062* t
<0.0001* |
<0.0001* 1
0.4502
Steel
0.3038
0.8211
<0.0001* f
<0.0001* f
1.0000
1.0000
<0.0001* f
<0.0001* f
Wood
<0.0001* |
<0.0001* 1
0.0426* t
0.4582
<0.0001* |
<0.0001* |
<0.0001* 1
0.9208
1.0000
<0.0001* |
Pure
25
75
7
Days
Paper
0.6700
1.0000
Plastic
0.0364* |
0.0933
0.8953
0.9897
Rubber
0.7215
0.4344
0.0149* f
1.0000
1.0000
0.6873
Steel
<0.0001* f
<0.0001* f
<0.0001* f
<0.0001* f
<0.0001* f
<0.0001* t
<0.0001* f
<0.0001* f
Wood
0.8187
0.8437
0.0614
0.5583
<0.0001* |
1.0000
1.0000
0.9643
1.0000
<0.0001* |
14
Days
Paper
0.7152
1.0000
Plastic
0.0001* |
<0.0001* |
0.0257* |
0.0076* |
Rubber
0.8187
0.5526
0.0003* t
1.0000
1.0000
0.0519
Steel
<0.0001* f
<0.0001* f
<0.0001* f
<0.0001* f
<0.0001* f
<0.0001* t
<0.0001* f
<0.0001* t
Wood
0.2418
0.1259
0.0065* t
0.3455
<0.0001* |
1.0000
0.9969
0.4612
1.0000
<0.0001* |
* Significant at the 0.05 level.
T, | "T" indicates the mean percent reduction of the row material is significantly greater than the column material, while "J," indicates the mean percent
reduction of the row material is significantly less than the column material.
B-17
-------
Table B-2f. Unadjusted and Tukey Adjusted P-values Comparing Materials for 30 °C and 45% Humidity, Crude and Pure
Ricin by Time.
Unadjusted P-Value
Tukey Adjusted P-Value
Ricin
Type
Temperature
(°C)
Humidity
(%)
Time
Material
Carpet
Paper
Plastic
Rubber
Steel
Carpet
Paper
Plastic
Rubber
Steel
Crude
30
45
7
Days
Paper
<0.0001* 1
0.0002* |
Plastic
0.4130
<0.0001* t
1.0000
<0.0001* t
Rubber
0.8722
<0.0001* t
0.3278
1.0000
0.0004* f
1.0000
Steel
0.0102* |
0.0093* t
0.0009* |
0.0157* |
0.5839
0.5581
0.1157
0.7022
Wood
0.0013* |
0.0518
<0.0001* |
0.0022* |
0.4949
0.1627
0.9468
0.0150* |
0.2351
1.0000
14
Days
Paper
Plastic
0.0064* |
0.3140
0.0793
0.4607
1.0000
0.9825
Rubber
0.0934
0.2780
0.4965
0.9898
1.0000
1.0000
Steel
0.2421
0.1110
0.8694
0.6059
1.0000
0.9947
1.0000
1.0000
Wood
0.0129* |
0.8029
0.1310
0.4026
0.1775
0.6473
1.0000
0.9974
1.0000
0.9995
Pure
30
45
7
Days
Paper
0.0045* t
0.3741
Plastic
<0.0001* |
<0.0001* |
<0.0001* |
<0.0001* |
Rubber
<0.0001* |
<0.0001* |
0.1455
0.0125* |
<0.0001* |
0.9985
Steel
<0.0001* |
<0.0001* |
0.6468
0.3160
0.0003* |
<0.0001* |
1.0000
1.0000
Wood
<0.0001* |
<0.0001* |
0.3199
0.6410
0.5903
0.0024* |
<0.0001* |
1.0000
1.0000
1.0000
14
Days
Paper
0.0098* f
0.5716
Plastic
0.0195* t
0.7949
0.7586
1.0000
Rubber
0.2600
0.1360
0.2170
1.0000
0.9979
0.9999
Steel
0.0187* t
0.8072
0.9872
0.2111
0.7481
1.0000
1.0000
0.9999
Wood
0.3786
0.0007* |
0.0015* |
0.0464* |
0.0015* |
1.0000
0.0940
0.1810
0.9330
0.1743
* Significant at the 0.05 level.
T, | "T" indicates the mean percent reduction of the row material is significantly greater than the column material, while "J," indicates the mean percent
reduction of the row material is significantly less than the column material.
B-18
-------
Table B-2g. Unadjusted and Tukey Adjusted P-values Comparing Materials for 30 °C and 70% Humidity, Crude and Pure
Ricin by Time.
Unadjusted P-Value
Tukey Adjusted P-Value
Ricin
Type
Temperature
(°C)
Humidity
(%)
Time
Material
Carpet
Paper
Plastic
Rubber
Steel
Carpet
Paper
Plastic
Rubber
Steel
Crude
30
75
7 Days
Paper
<0.0001* t
0.0002* t
Plastic
0.1409
0.0002* |
0.9982
0.0405* |
Rubber
0.5273
<0.0001* |
0.3972
1.0000
0.0022* |
1.0000
Steel
<0.0001* f
0.0186* t
<0.0001* f
<0.0001* t
<0.0001* f
0.7463
<0.0001* t
<0.0001* f
Wood
0.1630
0.0002* |
0.9372
0.4424
<0.0001* 1
0.9992
0.0318* |
1.0000
1.0000
<0.0001* 1
14
Days
Paper
0.2179
0.9999
Plastic
0.1955
0.0126* |
0.9997
0.6410
Rubber
Steel
0.0030* |
0.0001* f
<0.0001* |
0.0072* t
0.0855
<0.0001* f
<0.0001* f
0.2913
0.0240* t
0.0089* |
0.4908
0.9862
0.0002* t
<0.0001* f
Wood
0.0687
0.0027* |
0.5923
0.2330
<0.0001* 1
0.9734
0.2686
1.0000
0.9999
<0.0001* 1
Pure
30
75
7 Days
Paper
0.0238* t
0.8074
Plastic
0.0086* f
0.7013
0.5375
1.0000
Rubber
0.5534
0.0047* |
0.0015* |
1.0000
0.3858
0.1742
Steel
0.0243* t
0.9927
0.6945
0.0049* f
0.8127
1.0000
1.0000
0.3920
Wood
0.3293
0.0015* |
0.0004* |
0.7005
0.0015* |
1.0000
0.1738
0.0636
1.0000
0.1776
14
Days
Paper
0.8261
1.0000
Plastic
0.9419
0.7699
1.0000
1.0000
Rubber
0.5051
0.3761
0.5526
1.0000
1.0000
1.0000
Steel
<0.0001* f
<0.0001* f
<0.0001* f
<0.0001* f
<0.0001* f
<0.0001* f
<0.0001* f
<0.0001* f
Wood
<0.0001* |
<0.0001* |
<0.0001* |
0.0001* |
<0.0001* 1
0.0022* |
0.0009* |
0.0029* |
0.0228* |
<0.0001* |
* Significant at the 0.05 level.
T, | "T" indicates the mean percent reduction of the row material is significantly greater than the column material, while "J," indicates the mean percent
reduction of the row material is significantly less than the column material.
B-19
-------
Table B-3a. Unadjusted and Tukey Adjusted P-values Comparing Times for Crude
Ricin, by Temperature, Humidity, and Material.
Ricin
Type
Unadjusted P-
Value
Tukey Adjusted
P-Value
Temperature
(°C)
Humidity
(%)
Material
Day 7 vs. 14
Day 7 vs. 14
Carpet
0.3892
1.0000
Paper
0.4408
1.0000
20
75
Plastic
0.3233
1.0000
Rubber
0.4339
1.0000
Steel
0.0024* |
0.2481
Wood
0.3344
1.0000
Carpet
<0.0001*|
0.0034* |
Paper
0.5696
1.0000
45
Plastic
0.1970
0.9998
Rubber
0.0014*|
0.1666
Steel
0.9514
1.0000
25
Wood
0.5047
1.0000
Carpet
0.0593
0.9612
Paper
<0.0001* t
0.0184* t
75
Plastic
<0.0001* t
<0.0001* t
Crude
Rubber
0.9956
1.0000
Steel
0.0035* |
0.3218
Wood
0.0644
0.9684
Carpet
0.7378
1.0000
Paper
0.0057* |
0.4323
45
Plastic
0.1373
0.9980
Rubber
0.2340
0.9999
Steel
0.0784
0.9818
30
Wood
0.2715
1.0000
Carpet
0.1644
0.9992
Paper
0.0092* t
0.5547
75
Plastic
0.1686
0.9993
Rubber
0.0252* t
0.8205
Steel
0.0231* t
0.8003
Wood
0.0681
0.9727
40
20
Steel
0.8210
1.0000
* Significant at the 0.05 level.
T, | "T" indicates, for the specific column, that the mean percent reduction of the first time point is significantly
greater than the second time point, while "J," indicates, for the specific column, that the mean percent
reduction of the first time point is significantly less than the second time point. For example, the p-value and
arrow <0.0001* f for Paper in the Day 7 vs. 14 column would indicate that the mean percent reduction for
Paper on Day 7 was significantly greater than the mean percent reduction of Day 14.
B-20
-------
Table B-3b. Unadjusted and Tukey Adjusted P-values Comparing Times for Pure Ricin,
by Temperature, Humidity, and Material.
Ricin
Type
Unadjusted P-
Value
Tukey Adjusted
P-Value
Temperature
(°C)
Humidity
(%)
Material
Day 7 vs. 14
Day 7 vs. 14
Carpet
0.0065* |
0.4639
Paper
0.0014*|
0.1673
20
75
Plastic
<0.0001*|
0.0010*|
Rubber
0.0003* |
0.0420* |
Steel
0.0665
0.9709
Wood
<0.0001*|
0.0169*|
Carpet
<0.0001*|
<0.0001*|
Paper
<0.0001*|
0.0005* |
45
Plastic
0.0010*|
0.1340
Rubber
0.0003* |
0.0512
Steel
0.0038* |
0.3395
25
Wood
0.0020* |
0.2206
Carpet
0.0030* |
0.2859
Paper
0.0002* |
0.0372* |
Pure
75
Plastic
0.2292
0.9999
Rubber
0.0156*|
0.6993
Steel
0.0647
0.9687
Wood
0.0381*|
0.9033
Carpet
<0.0001* t
<0.0001* t
Paper
<0.0001* t
<0.0001* t
45
Plastic
0.1442
0.9984
Rubber
0.2187
0.9999
Steel
0.3062
1.0000
30
Wood
0.0064* t
0.4589
Carpet
0.4153
1.0000
Paper
0.0047* t
0.3836
70
Plastic
0.0006* t
0.0816
Rubber
0.3744
1.0000
Steel
<0.0001*|
<0.0001*|
Wood
<0.0001* t
0.0040* t
* Significant at the 0.05 level.
T, | "T" indicates, for the specific column, that the mean percent reduction of the first time point is significantly
greater than the second time point, while "J," indicates, for the specific column, that the mean percent
reduction of the first time point is significantly less than the second time point. For example, the p-value and
arrow <0.0001* f for Paper in the Day 7 vs. 14 column would indicate that the mean percent reduction for
Paper on Day 7 was significantly greater than the mean percent reduction of Day 14.
B-21
-------
Table B-3c. Unadjusted and Tukey Adjusted P-values Comparing Times for 20°C and 45% Humidity, by Ricin Type and
Material.
Unadjusted P-Value
Temperature (°C)
Humidity
(%)
Ricin
Type
Material
Day 7 vs. 14
Day 7 vs. 21
Day 7 vs. 28
Day 14 vs. 21
Day 14 vs. 28
Day 21 vs. 28
Carpet
0.8154
<0.0001* 1
<0.0001* 1
<0.0001* 1
<0.0001* 1
<0.0001* t
Paper
0.7571
0.0010* 1
<0.0001* 1
0.0027* |
<0.0001* 1
0.0896
Crude
Plastic
0.4152
0.6255
<0.0001* 1
0.1933
<0.0001* 1
<0.0001* |
Rubber
0.9616
0.6748
0.0003* 1
0.6400
0.0003* 1
0.0013* |
Steel
0.0096* |
0.0001* |
0.0001* 1
0.1812
0.2115
0.9296
20
45
Wood
0.1504
<0.0001* |
<0.0001* 1
0.0002* |
<0.0001* |
0.4027
Carpet
<0.0001* |
0.0536
<0.0001* 1
0.0002* t
0.2801
0.0082* |
Pure
Paper
Plastic
0.9252
0.0061* t
0.8463
0.1586
0.1718
0.8244
0.7736
0.1757
0.1444
0.0115* |
0.2404
0.2343
Rubber
0.0253* |
<0.0001* |
<0.0001* |
0.0081* |
0.0011* |
0.5192
Steel
<0.0001* |
0.0016* |
<0.0001* |
0.0165* t
0.0051* |
<0.0001* |
Wood
0.7103
0.0004* |
0.0381* |
<0.0001* |
0.0148* |
0.1235
Tukey Adjusted P-Value
Temperature (°C)
Humidity
(%)
Ricin
Type
Material
Day 7 vs. 14
Day 7 vs. 21
Day 7 vs. 28
Day 14 vs. 21
Day 14 vs. 28
Day 21 vs. 28
Carpet
1.0000
<0.0001* 1
0.0152* |
<0.0001* 1
0.0062* |
0.0580
Paper
1.0000
0.3307
0.0009* |
0.5749
0.0036* |
0.9999
Crude
Plastic
1.0000
1.0000
<0.0001* |
1.0000
<0.0001* |
<0.0001* |
Rubber
1.0000
1.0000
0.1519
1.0000
0.1330
0.4035
Steel
0.8783
0.0655
0.0862
1.0000
1.0000
1.0000
20
45
Wood
1.0000
0.0005* |
<0.0001* |
0.1150
0.0062* |
1.0000
Carpet
Paper
<0.0001* |
1.0000
0.9990
1.0000
0.0061* |
1.0000
0.1208
1.0000
1.0000
1.0000
0.8483
1.0000
Pure
Plastic
0.7840
1.0000
1.0000
1.0000
0.9077
1.0000
Rubber
0.9834
0.0016* |
<0.0001* |
0.8453
0.3511
1.0000
Steel
<0.0001* |
0.4424
<0.0001* 1
0.9528
0.7426
0.0004* |
Wood
1.0000
0.1681
0.9956
0.0559
0.9411
1.0000
" Significant at the 0.05 level.
f , I " f" indicates, for the specific column, that the mean percent reduction of the first time point is significantly greater than the second time point,
while " |" indicates, for the specific column, that the mean percent reduction of the first time point is significantly less than the second time point. For
example, the p-value and arrow <0.0001* f for Paper in the Day 7 vs. 14 column would indicate that the mean percent reduction for Paper on Day 7 was
significantly greater than the mean percent reduction of Day 14.
B-22
-------
Empty page????
B-23
-------
Table B-3d. Unadjusted P-values Comparing Times for 40 °C and 20% Humidity, for Steel by Ricin Type.
Unadjusted P-Value
Temperature
(°C)
Humidity
(%)
Ricin
Type
Time
48 Hours
72 Hours
96 Hours
120 Hours
144 Hours
7 Days
10 Days
11 Days
12 Days
13 Days
72 Hours
0.4751
96 Hours
0.6150
0.7952
120 Hours
0.8329
0.5373
0.7206
144 Hours
0.1409
0.0083* t
0.0167* t
0.0405* t
Crude
7 Days
0.2840
0.0304* t
0.0557
0.1178
0.6188
10 Days
0.6399
0.2111
0.2980
0.4529
0.3481
0.5936
11 Days
0.0088* f
0.0003* t
0.0005* t
0.0014* f
0.1120
0.0471* t
0.0298* t
40
20
12 Days
0.0271* t
0.0013* t
0.0026* t
0.0061* t
0.2709
0.1331
0.0790
0.6678
13 Days
0.0002* t
<0.0001* f
<0.0001* f
<0.0001* f
0.0043* t
0.0012* t
0.0012* t
0.2544
0.1181
14 Days
0.4632
0.1203
0.1787
0.2909
0.5273
0.7899
0.0555
0.1348
0.0027* |
72 Hours
0.5159
96 Hours
0.3048
0.7050
Pure
120 Hours
0.0606
0.2156
0.3881
144 Hours
0.0510
0.1885
0.3471
0.9382
7 Days
0.0603
0.2147
0.3868
0.9981
0.9400
* Significant at the 0.05 level.
T, | "T" indicates that the mean percent reduction of the row time point is significantly greater than the column time point, while " |" indicates that the mean
percent reduction of the row time point is significantly less than the column time point.
B-24
-------
Table B-3e. Tukey Adjusted P-values Comparing Times for 40 °C and 20% Humidity, for Steel by Ricin Type.
Tukey Adjusted P-Value
Temperature
(°C)
Humidity
(%)
Ricin
Type
Time
48 Hours
72 Hours
96 Hours
120 Hours
144 Hours
7 Days
10 Days
11 Days
12 Days
13 Days
72 Hours
1.0000
96 Hours
1.0000
1.0000
120 Hours
1.0000
1.0000
1.0000
144 Hours
0.9863
0.3731
0.5550
0.7989
Crude
7 Days
0.9996
0.7241
0.8718
0.9754
1.0000
10 Days
1.0000
0.9976
0.9997
1.0000
0.9999
1.0000
11 Days
0.3871
0.0236* t
0.0448* f
0.1002
0.9715
0.8352
0.7185
40
20
12 Days
0.6921
0.0981
0.1653
0.3071
0.9995
0.9833
0.9327
1.0000
13 Days
0.0225* t
0.0002* t
0.0005* t
0.0014* f
0.2395
0.0915
0.0875
0.9992
0.9756
14 Days
1.0000
0.9770
0.9946
0.9997
1.0000
1.0000
0.8710
0.9840
0.1696
72 Hours
1.0000
96 Hours
0.9998
1.0000
Pure
120 Hours
0.8886
0.9979
1.0000
144 Hours
0.8532
0.9958
0.9999
1.0000
7 Days
0.8876
0.9978
1.0000
1.0000
1.0000
* Significant at the 0.05 level.
T, | "T" indicates that the mean percent reduction of the row time point is significantly greater than the column time point, while "J," indicates that the mean
percent reduction of the row time point is significantly less than the column time point.
B-25
-------
Table B-3f. Unadjusted P-values Comparing Times for 50 °C and 20% Humidity, for Steel by Ricin Type.
Unadjusted P-Value
Temperature
(°C)
Humidity
(%)
Ricin
Type
Time
6 Hours
24 Hours
30 Hours
48 Hours
72 Hours
96 Hours
120 Hours
144 Hours
24 Hours
0.0486* f
30 Hours
0.7326
0.0213* |
48 Hours
0.4290
0.1339
0.2369
Crude
72 Hours
0.5320
0.0964
0.3089
0.8383
96 Hours
0.0526
0.7314
0.0204* t
0.1561
0.1055
120 Hours
0.1803
0.5191
0.0938
0.4469
0.3542
0.6878
144 Hours
0.4071
0.2473
0.2427
0.8675
0.7389
0.3200
0.6066
50
20
7 Days
<0.0001* f
<0.0001* f
<0.0001* f
<0.0001* f
<0.0001* f
<0.0001* f
<0.0001* f
<0.0001* f
24 Hours
0.0037* t
30 Hours
<0.0001* f
0.1812
Pure
48 Hours
72 Hours
<0.0001* f
<0.0001* f
<0.0001* f
<0.0001* f
0.0134* t
<0.0001* f
0.0003* t
96 Hours
<0.0001* f
<0.0001* f
<0.0001* f
<0.0001* f
0.1825
120 Hours
<0.0001* f
<0.0001* f
<0.0001* f
<0.0001* f
0.0072* t
0.1022
144 Hours
<0.0001* f
<0.0001* f
<0.0001* f
<0.0001* f
0.0042* t
0.0692
0.8713
7 Days
<0.0001* f
<0.0001* f
<0.0001* f
<0.0001* f
<0.0001* f
<0.0001* f
<0.0001* f
<0.0001* t
* Significant at the 0.05 level.
T, | "T" indicates that the mean percent reduction of the row time point is significantly greater than the column time point, while "J," indicates that the mean
percent reduction of the row time point is significantly less than the column time point.
B-26
-------
Table B-3g. Tukey Adjusted P-values Comparing Times for 50 °C and 20% Humidity, for Steel by Ricin Type.
Tukey Adjusted P-Value
Temperature
(°C)
Humidity
(%)
Ricin
Type
Time
6 Hours
24 Hours
30 Hours
48 Hours
72 Hours
96 Hours
120 Hours
144 Hours
24 Hours
0.8641
30 Hours
48 Hours
1.0000
1.0000
0.6539
0.9880
0.9992
Crude
72 Hours
1.0000
0.9665
0.9999
1.0000
96 Hours
0.8803
1.0000
0.6412
0.9933
0.9739
120 Hours
0.9965
1.0000
0.9640
1.0000
1.0000
1.0000
144 Hours
1.0000
0.9994
0.9993
1.0000
1.0000
0.9999
1.0000
50
20
7 Days
<0.0001* f
<0.0001* f
<0.0001* f
<0.0001* f
<0.0001* f
<0.0001* f
<0.0001* f
<0.0001* t
24 Hours
0.2357
30 Hours
0.0045* t
0.9966
48 Hours
<0.0001* f
0.0106* t
0.5238
Pure
72 Hours
<0.0001* f
<0.0001* f
<0.0001* f
0.0334* t
96 Hours
<0.0001* f
<0.0001* f
<0.0001* f
0.0003* t
0.9967
120 Hours
<0.0001* f
<0.0001* f
<0.0001* f
<0.0001* f
0.3643
0.9715
144 Hours
<0.0001* f
<0.0001* f
<0.0001* f
<0.0001* f
0.2539
0.9272
1.0000
7 Days
<0.0001* f
<0.0001* f
<0.0001* f
<0.0001* f
<0.0001* f
<0.0001* f
<0.0001* f
<0.0001* f
* Significant at the 0.05 level.
T, | "T" indicates the mean percent reduction of the row time point is significantly greater than the column time point, while "J," indicates the mean percent
reduction of the row time point is significantly less than the column time point.
B-27
-------
Table B-4a. Unadjusted and Tukey Adjusted P-values Comparing Ricin Types, for 20 °C
and 45% Humidity, by Time and Material.
Unadjusted P-
Value
Tukey Adjusted
P-Value
Temperature
(°C)
Humidity
(%)
Time
Material
Crude vs. Pure
Crude vs. Pure
Carpet
0.0574
0.9993
Paper
0.0540
0.9990
7 Days
Plastic
<0.0001*|
<0.0001*|
Rubber
0.6237
1.0000
Steel
<0.0001*|
0.0015*|
Wood
0.0440* |
0.9975
Carpet
<0.0001*|
<0.0001*|
Paper
0.1264
1.0000
14 Days
Plastic
<0.0001*|
0.0006* |
Rubber
0.0057* |
0.7710
Steel
<0.0001*|
<0.0001*|
20
45
Wood
0.8327
1.0000
Carpet
<0.0001* t
0.0088* t
Paper
0.2243
1.0000
21 Days
Plastic
<0.0001*|
0.0004* |
Rubber
<0.0001*|
0.0012*|
Steel
<0.0001*|
0.0296* |
Wood
0.6558
1.0000
Carpet
0.0331*|
0.9927
Paper
0.0822
0.9999
28 Days
Plastic
0.5590
1.0000
Rubber
0.0176*|
0.9589
Steel
<0.0001*|
<0.0001*|
Wood
0.0539
0.9990
* Significant at the 0.05 level.
T, | "T" indicates, for the specific column, that the mean percent reduction of the first ricin type is significantly
greater than the mean percent reduction of the second ricin type, while "J," indicates, for the specific column,
that the mean percent reduction of the first ricin type is significantly less than the mean percent reduction of
the second ricin type. For example, the p-value and arrow <0.0001* f for Paper in the Crude vs. Pure
column would indicate that the mean percent reduction for Crude Paper was significantly greater than the
mean percent reduction of the Pure.
B-28
-------
Table B-4b. Unadjusted and Tukey Adjusted P-values Comparing Ricin Types, for 20 °C
and 75% Humidity, by Time and Material.
Unadjusted P-
Value
Tukey Adjusted
P-Value
Temperature
(°C)
Humidity
(%)
Time
Material
Crude vs. Pure
Crude vs. Pure
Carpet
Paper
0.6282
0.0096* t
1.0000
0.5665
7 Days
Plastic
0.0018* t
0.2037
Rubber
Steel
0.2666
<0.0001*|
1.0000
<0.0001*|
20
75
Wood
<0.0001* t
<0.0001* t
Carpet
Paper
0.1558
0.1575
0.9989
0.9990
14 Days
Plastic
0.0089* |
0.5453
Rubber
Steel
0.0603
<0.0001*|
0.9627
0.0001*|
Wood
<0.0001* t
<0.0001* t
* Significant at the 0.05 level.
T, | "T" indicates, for the specific column, that the mean percent reduction of the first ricin type is significantly
greater than the mean percent reduction of the second ricin type, while "J," indicates, for the specific column,
that the mean percent reduction of the first ricin type is significantly less than the mean percent reduction of
the second ricin type. For example, the p-value and arrow <0.0001* f for Paper in the Crude vs. Pure
column would indicate that the mean percent reduction for Crude Paper was significantly greater than the
mean percent reduction of the Pure.
Table B-4c. Unadjusted and Tukey Adjusted P-values Comparing Ricin Types, for 25 °C
and 45% Humidity, by Time and Material.
B-29
-------
Unadjusted P-
Value
Tukey
Adjusted P-
Value
Temperature
(°C)
Humidity
(%)
Time
Material
Crude vs. Pure
Crude vs. Pure
Carpet
Paper
0.0524
<0.0001* t
0.9481
0.0063* t
7 Days
Plastic
0.0159*|
0.7048
Rubber
Steel
0.1604
0.0001*|
0.9991
0.0258* |
25
45
Wood
0.0253* t
0.8218
Carpet
Paper
0.0010*|
0.8988
0.1342
1.0000
14 Days
Plastic
<0.0001*|
0.0035* |
Rubber
Steel
0.3330
<0.0001*|
1.0000
<0.0001*|
Wood
0.8176
1.0000
* Significant at the 0.05 level.
T, | "T" indicates, for the specific column, that the mean percent reduction of the first ricin type is significantly
greater than the mean percent reduction of the second ricin type, while "J," indicates, for the specific column,
that the mean percent reduction of the first ricin type is significantly less than the mean percent reduction of
the second ricin type. For example, the p-value and arrow <0.0001* f for Paper in the Crude vs. Pure
column would indicate that the mean percent reduction for Crude Paper was significantly greater than the
mean percent reduction of the Pure.
B-30
-------
Table B-4d. Unadjusted and Tukey Adjusted P-values Comparing Ricin Types, for 25 °C
and 75% Humidity, by Time and Material.
Unadjusted P-
Value
Tukey Adjusted
P-Value
Temperature
(°C)
Humidity
(%)
Time
Material
Crude vs. Pure
Crude vs. Pure
Carpet
0.0027* t
0.2662
Paper
<0.0001* t
<0.0001* t
7 Days
Plastic
0.0018* t
0.2034
Rubber
0.0063* |
0.4571
Steel
<0.0001*|
<0.0001*|
25
75
Wood
0.2865
1.0000
Carpet
0.0549
0.9533
Paper
0.4430
1.0000
14 Days
Plastic
<0.0001*|
0.0079* |
Rubber
<0.0001*|
0.0002* |
Steel
<0.0001*|
<0.0001*|
Wood
<0.0001*|
0.0005* |
* Significant at the 0.05 level.
T, | "T" indicates, for the specific column, that the mean percent reduction of the first ricin type is significantly
greater than the mean percent reduction of the second ricin type, while "J," indicates, for the specific column,
that the mean percent reduction of the first ricin type is significantly less than the mean percent reduction of
the second ricin type. For example, the p-value and arrow <0.0001* f for Paper in the Crude vs. Pure
column would indicate that the mean percent reduction for Crude Paper was significantly greater than the
mean percent reduction of Pure.
B-31
-------
Table B-4e. Unadjusted and Tukey Adjusted P-values Comparing Ricin Types, for 30 °C
and 45% Humidity, by Time and Material.
Unadjusted P-
Value
Tukey
Adjusted P-
Value
Temperature
(°C)
Humidity
(%)
Time
Material
Crude vs. Pure
Crude vs. Pure
Carpet
0.0001*1
0.0231*|
Paper
<0.0001*1
<0.0001*|
7 Days
Plastic
0.0150* t
0.6900
Rubber
0.9800
1.0000
Steel
0.1572
0.9990
30
45
Wood
0.0094* |
0.5606
Carpet
0.0047* t
0.3848
Paper
0.0131*|
0.6521
14 Days
Plastic
0.6221
1.0000
Rubber
0.9477
1.0000
Steel
0.5011
1.0000
Wood
0.2170
0.9999
* Significant at the 0.05 level.
T, | "T" indicates, for the specific column, that the mean percent reduction of the first ricin type is significantly
greater than the mean percent reduction of the second ricin type, while "J," indicates, for the specific column,
that the mean percent reduction of the first ricin type is significantly less than the mean percent reduction of
the second ricin type. For example, the p-value and arrow <0.0001* f for Paper in the Crude vs. Pure
column would indicate that the mean percent reduction for Crude Paper was significantly greater than the
mean percent reduction of Pure.
B-32
-------
Table B-4f. Unadjusted and Tukey Adjusted P-values Comparing Ricin Types, for 30 °C
and 70% Humidity, by Time and Material.
Unadjusted P-
Value
Tukey
Adjusted P-
Value
Temperature
(°C)
Humidity
(%)
Time
Material
Crude vs. Pure
Crude vs. Pure
Carpet
0.0021*|
0.2232
Paper
0.8694
1.0000
7 Days
Plastic
<0.0001*|
0.0067* |
Rubber
0.0555
0.9544
Steel
0.0275* t
0.8400
30
70
Wood
0.4363
1.0000
Carpet
0.3452
1.0000
Paper
0.9433
1.0000
14 Days
Plastic
0.0318*|
0.8701
Rubber
0.0013*|
0.1568
Steel
<0.0001*|
<0.0001*|
Wood
0.0632
0.9668
* Significant at the 0.05 level.
T, | "T" indicates, for the specific column, the mean percent reduction of the first ricin type is significantly greater
than the mean percent reduction of the second ricin type, while "J," indicates, for the specific column, that the
mean percent reduction of the first ricin type is significantly less than the mean percent reduction of the
second ricin type. For example, the p-value and arrow <0.0001* f for Paper in the Crude vs. Pure column
would indicate that the mean percent reduction for Crude Paper was significantly greater than the mean
percent reduction of Pure.
B-33
-------
Table B-4g. Unadjusted and Tukey Adjusted P-values Comparing Ricin Types, for Steel
at 40 °C and 20% Humidity, by Time.
Unadjusted P-
Value
Tukey
Adjusted P-
Value
Temperature
(°C)
Humidity
(%)
Time
Material
Crude vs. Pure
Crude vs. Pure
40
20
48 Hours
Steel
0.0005* |
0.0450* |
72 Hours
Steel
<0.0001*|
<0.0001*|
96 Hours
Steel
<0.0001*|
<0.0001*|
120 Hours
Steel
<0.0001*1
<0.0001*|
144 Hours
Steel
<0.0001*1
0.0004* |
7 Days
Steel
<0.0001*1
0.0001*|
Significant at the 0.05 level.
T, | "T" indicates, for the specific column, that the mean percent reduction of the first ricin type is significantly
greater than the mean percent reduction of the second ricin type, while "J," indicates, for the specific column,
that the mean percent reduction of the first ricin type is significantly less than the mean percent reduction of
the second ricin type. For example, the p-value and arrow <0.0001* f for Paper in the Crude vs. Pure
column would indicate that the mean percent reduction for Crude Paper was significantly greater than the
mean percent reduction of Pure.
Table B-4h. Unadjusted and Tukey Adjusted P-values Comparing Ricin Types, for Steel
at 50 °C and 20% Humidity, by Time.
Unadjusted P-
Value
Tukey
Adjusted P-
Value
Temperature
(°C)
Humidity
(%)
Time
Material
Crude vs. Pure
Crude vs. Pure
50
20
6 Hours
Steel
0.1163
0.9806
24 Hours
Steel
0.0121*|
0.4950
30 Hours
Steel
<0.0001*|
<0.0001*|
48 Hours
Steel
<0.0001*|
<0.0001*|
72 Hours
Steel
<0.0001*|
<0.0001*|
96 Hours
Steel
<0.0001*|
<0.0001*|
120 Hours
Steel
<0.0001*|
<0.0001*|
144 Hours
Steel
<0.0001*1
<0.0001*|
7 Days
Steel
<0.0001*1
<0.0001*1
Significant at the 0.05 level.
T, | "T" indicates, for the specific column, that the mean percent reduction of the first ricin type is significantly
greater than the mean percent reduction of the second ricin type, while "J," indicates, for the specific column,
that the mean percent reduction of the first ricin type is significantly less than the mean percent reduction of
the second ricin type. For example, the p-value and arrow <0.0001* f for Paper in the Crude vs. Pure
column would indicate that the mean percent reduction for Crude Paper was significantly greater than the
mean percent reduction of Pure.
B-34
-------
Table B-5a. Unadjusted and Tukey Adjusted P-values Comparing Humidity, for Crude
Ricin, by Material, Time and Temperature.
Ricin Type
Unadjusted P-
Value
Tukey Adjusted
P-Value
Material
Time
Temperature
(°C)
45% vs. 75%
45% vs. 75%
20
0.3652
0.9986
7 Days
25
<0.0001* |
0.0003* |
Carpet
30
<0.0001* f
0.0038* t
20
0.1656
0.9562
14 Days
25
0.0010* |
0.0409* |
30
0.0011* f
0.0460* f
20
0.2180
0.9817
7 Days
25
0.0002* |
0.0085* |
Paper
30
<0.0001* |
<0.0001* |
20
0.4451
0.9997
14 Days
25
0.5801
1.0000
30
0.5053
0.9999
20
0.1092
0.8882
7 Days
25
0.0189* |
0.4068
Plastic
30
<0.0001* f
0.0003* t
20
0.0817
0.8213
14 Days
25
<0.0001* f
0.0001* f
Crude
30
<0.0001* f
0.0020* t
20
0.2906
0.9947
7 Days
25
0.1361
0.9287
Rubber
30
<0.0001* f
0.0004* f
20
0.1273
0.9176
14 Days
25
<0.0001* f
0.0011* f
30
<0.0001* f
<0.0001* f
20
<0.0001* |
<0.0001* |
7 Days
25
0.0012* |
0.0697
Steel
30
<0.0001* |
<0.0001* |
20
<0.0001* |
<0.0001* |
14 Days
25
<0.0001* 1
<0.0001* |
30
0.0330* |
0.6786
20
<0.0001* |
0.0031* |
7 Days
25
0.9279
1.0000
Wood
30
0.4648
0.9998
20
0.0005* |
0.0231* |
14 Days
25
0.0139* t
0.3333
30
0.0036* t
0.1226
* Significant at the 0.05 level.
T, | "T" indicates, for the specific column, that the mean percent reduction for the first humidity is significantly
greater than the mean percent reduction for the second humidity, while "J," indicates, for the specific column,
the mean percent reduction for the first humidity is significantly less than the mean percent reduction for the
second humidity. For example, the p-value and arrow <0.0001* f for Paper in the 45% vs. 75% column
would indicate that the mean percent reduction for Paper at 45% humidity was significantly greater than the
mean percent reduction for 75% humidity.
B-35
-------
Table B-5b. Unadjusted and Tukey Adjusted P-values Comparing Humidity, for Pure
Ricin, by Material, Time and Temperature.
Ricin Type
Unadjusted P-
Value
Tukey Adjusted
P-Value
Material
Time
Temperature
(°C)
45% vs. 75%
45% vs. 75%
20
0.5020
0.9999
7 Days
25
0.0836
0.8272
Carpet
30
<0.0001* f
<0.0001* f
20
0.0001* f
0.0068* f
14 Days
25
0.5405
1.0000
30
0.4248
0.9996
20
0.1482
0.9417
7 Days
25
0.6636
1.0000
Paper
30
<0.0001* f
<0.0001* f
20
0.5186
0.9999
14 Days
25
0.8445
1.0000
30
0.0019* f
0.0716
20
<0.0001* f
<0.0001* f
7 Days
25
<0.0001* f
0.0002* t
Plastic
30
<0.0001* |
<0.0001* |
20
0.0974
0.8636
14 Days
25
<0.0001* f
<0.0001* f
Pure
30
<0.0001* f
0.0024* t
20
0.9864
1.0000
7 Days
25
<0.0001* |
0.0030* |
Rubber
30
0.0005* t
0.0212* t
20
0.8006
1.0000
14 Days
25
0.0025* |
0.0902
30
0.0127* t
0.3135
20
0.0012* |
0.0613
7 Days
25
<0.0001* |
<0.0001* |
Steel
30
0.1215
0.9460
20
0.3052
0.9987
14 Days
25
<0.0001* |
<0.0001* |
30
<0.0001* |
<0.0001* |
20
0.0042* t
0.1386
7 Days
25
0.0003* |
0.0143* |
Wood
30
0.0130* t
0.3188
20
0.9085
1.0000
14 Days
25
0.0031* |
0.1073
30
0.0002* t
0.0102* t
* Significant at the 0.05 level.
T, | "T" indicates, for the specific column, that the mean percent reduction for the first humidity is significantly
greater than the mean percent reduction for the second humidity, while "J," indicates, for the specific column,
that the mean percent reduction for the first humidity is significantly less than the mean percent reduction for
the second humidity. For example, the p-value and arrow <0.0001* f for Paper in the 45% vs. 75% column
would indicate that the mean percent reduction for Paper at 45% humidity was significantly greater than the
mean percent reduction at 75% humidity.
B-36
-------
Table B-6a. Unadjusted and Tukey Adjusted P-values Comparing Temperatures, for
Carpet by Ricin Type, Humidity and Time.
Unadjusted P-Value
Tukey Adjusted P-
Value
Ricin
Type
Material
Humidity
(%)
Time
Temperature
(°C)
20 °C
25 °C
20 °C
25 °C
7
Days
14
Days
25
0.7786
1.0000
45
30
25
<0.0001*
t
0.0038* t
<0.0001*
t
0.0021* t
0.1281
0.0008* t
30
<0.0001*
t
0.0502
0.0004* t
0.6851
Crude
Carpet
7
Days
25
0.0003* t
0.0126* t
30
0.4741
<0.0001*
1
0.9999
0.0014*|
75
14
25
<0.0001*
t
0.0003* t
Days
30
0.8611
<0.0001*
1
1.0000
0.0005* |
7
Days
25
0.8795
1.0000
45
30
<0.0001*
t
<0.0001*
t
<0.0001*
t
<0.0001*
t
14
Days
25
0.4876
0.9999
Pure
Carpet
30
<0.0001*
1
<0.0001*
1
0.0040* |
0.0004* |
7
25
0.0264* t
0.4967
75
Days
30
0.0022* t
0.3476
0.0810
0.9981
14
25
0.0001* t
0.0053* t
Days
30
0.5325
0.0008* |
1.0000
0.0324* |
* Significant at the 0.05 level.
T, | "T" indicates that the mean percent reduction of the row temperature is significantly greater than the mean
percent reduction of the column temperature, while "J," indicates the mean percent reduction of the row
temperature is significantly less than the mean percent reduction of the column temperature.
B-37
-------
Table B-6b. Unadjusted and Tukey Adjusted P-values Comparing Temperatures, for
Paper by Ricin Type, Humidity and Time.
Unadjusted P-
Value
Tukey Ad
Va
justed P-
ue
Ricin
Type
Material
Humidity
(%)
Time
Temperature
(°C)
20 °C
25 °C
20 °C
25 °C
Crude
Paper
45
7
Days
14
Days
25
<0.0001*
t
0.0020* t
30
25
0.2415
<0.0001*
t
<0.0001*
1
0.9877
0.0011* t
<0.0001*
1
30
0.1200
0.0028* |
0.9069
0.0991
75
7
Days
14
Days
25
<0.0001*
t
<0.0001*
t
30
25
0.0013* t
<0.0001*
t
0.0003* |
0.0508
0.0021* t
0.0125*|
30
0.1444
0.0038* |
0.9378
0.1285
Pure
Paper
45
7
Days
25
0.4256
0.9996
30
<0.0001*
t
<0.0001*
t
<0.0001*
t
<0.0001*
t
14
Days
25
<0.0001*
t
<0.0001*
t
30
<0.0001*
t
0.3394
0.0001* t
0.9978
75
7
Days
25
0.0093* t
0.2523
30
<0.0001*
t
<0.0001*
t
<0.0001*
t
0.0012* t
14
Days
25
<0.0001*
t
<0.0001*
t
30
0.1596
0.0002* |
0.9517
0.0089* |
* Significant at the 0.05 level.
T, | "T" indicates that the mean percent reduction of the row temperature is significantly greater than the mean
percent reduction of the column temperature, while "J," indicates that the mean percent reduction of the row
temperature is significantly less than the mean percent reduction of the column temperature.
B-38
-------
Table B-6c. Unadjusted and Tukey Adjusted P-values Comparing Temperatures, for
Plastic by Ricin Type, Humidity and Time.
Unadjusted P-Value
Tukey Ad
Va
justed P-
ue
Ricin
Type
Material
Humidity
(%)
Time
Temperature
(°C)
20 °C
25 °C
20 °C
25 °C
Crude
Plastic
45
7
Days
25
30
0.0784
<0.0001*
t
<0.0001*
t
0.8107
<0.0001*
t
0.0002* t
14
Days
25
0.0007* t
0.0291* t
30
<0.0001*
t
0.0529
<0.0001*
t
0.7006
75
7
Days
25
30
0.0125* t
0.7086
0.0311*|
0.3101
1.0000
0.5437
14
Days
25
0.0008* |
0.0357* |
30
0.4837
0.0063* t
0.9999
0.1896
Pure
Plastic
45
7
Days
25
0.0057* |
0.1758
30
0.0006* |
0.4543
0.0284* |
0.9998
14
Days
25
<0.0001*
t
<0.0001*
t
30
0.0011* t
0.0247* |
0.0460* t
0.4775
75
7
Days
25
0.1600
0.9520
30
<0.0001*
t
<0.0001*
t
<0.0001*
t
<0.0001*
t
14
Days
25
0.5249
1.0000
30
0.5028
0.1946
0.9999
0.9729
* Significant at the 0.05 level.
T, | "T" indicates that the mean percent reduction of the row temperature is significantly greater than the mean
percent reduction of the column temperature, while "J," indicates that the mean percent reduction of the row
temperature is significantly less than the mean percent reduction of the column temperature.
B-39
-------
Table B-6d. Unadjusted and Tukey Adjusted P-values Comparing Temperatures, for
Rubber by Ricin Type, Humidity and Time.
Unadjusted P-Value
Tukey Ad
Va
justed P-
ue
Ricin
Type
Material
Humidity
(%)
Time
Temperature
(°C)
20 °C
25 °C
20 °C
25 °C
Crude
Rubber
45
7
Days
25
30
0.0569
<0.0001*
t
0.0002* t
0.7223
<0.0001*
t
0.0085* |
14
Days
25
<0.0001*
t
0.0002* t
30
0.0001* t
0.3282
0.0055* |
0.9973
75
7
Days
25
30
0.5293
0.9424
0.5773
1.0000
1.0000
1.0000
14
Days
25
0.2907
0.9947
30
0.0016*|
0.0279* |
0.0639
0.5119
Pure
Rubber
45
7
Days
25
0.8389
1.0000
30
<0.0001*
t
<0.0001*
t
<0.0001*
t
<0.0001*
t
14
Days
25
0.0112* t
0.2879
30
0.0186* t
0.8397
0.4035
1.0000
75
7
Days
25
<0.0001*
t
0.0016* t
30
<0.0001*
t
0.7626
0.0043* t
1.0000
14
Days
25
<0.0001*
t
<0.0001*
t
30
0.6845
<0.0001*
1
1.0000
<0.0001*
1
* Significant at the 0.05 level.
T, | "T" indicates that the mean percent reduction of the row temperature is significantly greater than the mean
percent reduction of the column temperature, while "J," indicates that the mean percent reduction of the row
temperature is significantly less than the mean percent reduction of the column temperature.
B-40
-------
Table B-6e. Unadjusted and Tukey Adjusted P-values Comparing Temperatures, for
Steel by Ricin Type, Humidity and Time.
Unadjusted P-Value
Tukey Ad
Va
justed P-
ue
Ricin
Type
Material
Humidity
(%)
Time
Temperature
(°C)
20 °C
25 °C
20 °C
25 °C
Crude
Steel
45
7
Days
25
0.0136* t
0.4299
30
0.0007* t
0.3004
0.0429* t
0.9992
14
Days
25
0.8965
1.0000
30
0.0001* t
<0.0001*
t
0.0092* t
0.0061* t
75
7
Days
25
30
0.3044
<0.0001*
t
<0.0001*
t
0.9992
0.0002* t
<0.0001*
t
14
Days
25
0.4017
0.9999
30
0.9315
0.3556
1.0000
0.9998
Pure
Steel
45
7
Days
25
0.2802
0.9977
30
0.5321
0.6460
1.0000
1.0000
14
Days
25
0.8727
1.0000
30
0.1589
0.2105
0.9743
0.9907
75
7
Days
25
<0.0001*
t
0.0059* |
30
0.2254
<0.0001*
1
0.9931
<0.0001*
1
14
Days
25
<0.0001*
t
0.0006* t
30
0.0003* t
0.2929
0.0193* t
0.9983
* Significant at the 0.05 level.
T, | "T" indicates that the mean percent reduction of the row temperature is significantly greater than the mean
percent reduction of the column temperature, while "J," indicates that the mean percent reduction of the row
temperature is significantly less than the mean percent reduction of the column temperature.
B-41
-------
Table B-6f. Unadjusted and Tukey Adjusted P-values Comparing Temperatures, for
Wood by Ricin Type, Humidity and Time.
Unadjusted P-Value
Tukey Adjusted P-
Value
Ricin
Type
Material
Humidity
(%)
Time
Temperature
(°C)
20 °C
25 °C
20 °C
25 °C
Crude
Wood
45
7
Days
25
0.0007* t
0.0309* t
30
0.0025* t
0.6716
0.0911
1.0000
14
Days
25
0.0039* t
0.1293
30
0.0007* t
0.5695
0.0315* t
1.0000
75
7
Days
25
0.4874
0.9999
30
0.6330
0.8273
1.0000
1.0000
14
Days
25
0.0022* |
0.0806
30
0.0026* |
0.9475
0.0943
1.0000
Pure
Wood
45
7
Days
25
0.8386
1.0000
30
<0.0001*
t
<0.0001*
t
<0.0001*
t
<0.0001*
t
14
Days
25
0.0008* t
0.0354* t
30
0.0675
0.0963
0.7707
0.8611
75
7
Days
25
<0.0001*
t
<0.0001*
t
30
<0.0001*
t
0.9832
<0.0001*
t
1.0000
14
Days
25
<0.0001*
t
<0.0001*
t
30
0.0487* |
<0.0001*
1
0.6761
<0.0001*
1
* Significant at the 0.05 level.
T, | "T" indicates that the mean percent reduction of the row temperature is significantly greater than the mean
percent reduction of the column temperature, while "J," indicates the mean percent reduction of the row
temperature is significantly less than the mean percent reduction of the column temperature.
B-42
-------
Table B-6g. Unadjusted and Tukey Adjusted P-values Comparing 40°C and 50°C, for
Steel by Ricin Type, Humidity and Time.
Unadjusted
P-Value
Tukey
Adjusted
P-Value
Ricin
Type
Material
Humidity
(%)
Time
Temperature
(°C)
o
o
o
40 °C
Crude
Steel
20
48 Hours
50
0.2126
0.9985
72 Hours
50
0.0103* t
0.4554
96 Hours
50
0.0006* t
0.0575
120 Hours
50
0.0338* t
0.7811
144 Hours
50
0.4834
1.0000
7 Days
50
<0.0001* t
<0.0001* t
Pure
Steel
20
48 Hours
50
0.0019* t
0.1419
72 Hours
50
<0.0001* t
0.0002* t
96 Hours
50
<0.0001* t
<0.0001* t
120 Hours
50
<0.0001* t
0.0014* t
144 Hours
50
<0.0001* t
0.0013* t
7 Days
50
<0.0001* t
<0.0001* t
* Significant at the 0.05 level.
T, | "T" indicates that the mean percent reduction of the row temperature is significantly greater than the mean
percent reduction of the column temperature, while "J," indicates the mean percent reduction of the row
temperature is significantly less than the mean percent reduction of the column temperature.
B-43
-------
Table B-7a. Unadjusted and Tukey Adjusted P-values Comparing Temperatures, for
Steel by Ricin Type at Day 7 with Relative Humidity of 20% or 45%.
Unadjusted P-Value
Tukey Adjusted
P-Value
Ricin
Type
Time
Temperature
(°C)
O
o
O
CM
25 °C
40 °C
O
o
O
CM
25 °C
o
o
o
Crude
7 Days
25
0.0337*
t
0.4261
30
0.0035*
t
0.3713
0.0753
0.9915
40
0.0020*
t
0.4448
0.0457*
t
0.9971
50
<0.0001*
t
<0.0001*
t
<0.0001*
t
<0.0001*
t
<0.0001*
t
<0.0001*
t
Pure
7 Days
25
0.0813
0.6247
30
0.3072
0.4516
0.9647
0.9940
40
<0.0001*
t
0.0047*
t
0.0008*
t
0.0789
50
<0.0001*
t
<0.0001*
t
<0.0001*
t
<0.0001*
t
<0.0001*
t
<0.0001*
t
* Significant at the 0.05 level.
T, | "T" indicates that the mean percent reduction of the row temperature is significantly greater than the mean
percent reduction of the column temperature, while "J," indicates that the mean percent reduction of the row
temperature is significantly less than the mean percent reduction of the column temperature.
Table B-7b. Unadjusted and Tukey Adjusted P-values Comparing Temperatures, for
Steel by Ricin Type at Day 14 with Relative Humidity of 20% or 45%.
Unadjusted P-Value
Tukey Adjusted P-Value
Ricin
Type
Time
Temperature
(°C)
O
o
O
CM
25 °C
O
o
O
CM
25 °C
Crude
14 Days
25
0.9105
1.0000
30
0.0010* t
0.0007* t
0.0248* t
0.0184* t
40
0.7265
0.6442
1.0000
0.9999
Pure
14 Days
25
0.7922
1.0000
30
0.0247* |
0.0446* |
0.2961
0.4417
* Significant at the 0.05 level.
T, | "T" indicates that the mean percent reduction of the row temperature is significantly greater than the mean
percent reduction of the column temperature, while "J," indicates that the mean percent reduction of the row
temperature is significantly less than the mean percent reduction of the column temperature.
B-44
-------
Temperature=2Q°C. Humidity=45%
100
C
o
o
¦D -100
Q)
Dd
| -200
O
d)
Q.
-300
-400
Ch-
w-
A
^ m
©¦
ct
o
TI
W. _
&L
r- ' O
o
A
OJ- ^
0
~
~
Carpet Paper Plastic Rubber
Material
Steel
Wood
O Crude, 7 Days 0 Pure, 7 Days A Crude, 14 Days ~ Pure, 14 Days
Figure B-1a. Geometric Mean Percent Reduction, 95% Confidence Intervals, and
Observed Percent Reduction Plotted by Material and Ricin Type for Days 7
and 14, 20 °C and 45% Humidity.
Temperature=20<'C, Humidity=45%
100
C
o
O 0!
Z5
T3
0)
Od
S -100
CD
Q.
-200
&
o-
A
o
©f
o
« jaf
©t- "A
~
Carpet
Paper
Steel
Wood
Plastic Rubber
Material
O Crude, 21 Days 0 Pure, 21 Days A Crude, 28 Days LI Pure, 28 Days
Figure B-1b. Geometric Mean Percent Reduction, 95% Confidence Intervals, and
Observed Percent Reduction Plotted by Material and Ricin Type for Days 21
and 28, 20 °C and 45% Humidity.
B-45
-------
Temperature=20°C, Humidity=75%
C
o
o
ZJ
"O
Q)
Od
¦+—'
C
s
u-
0
Q.
100-
0
-100
-200
-300
-400
-500
-600
-700
Q^tY- -
0
f
O 1
1
O
Carpet Paper Plastic Rubber
Material
Steel
Wood
O Crude, 7 Days 0 Pure, 7 Days A Crude, 14 Days Pure, 14 Days
Figure B-1c. Geometric Mean Percent Reduction, 95% Confidence Intervals, and
Observed Percent Reduction Plotted by Material and Ricin Type for Days 7
and 14, 20 °C and 75% Humidity.
c
o
o
TJ
0)
Dd
C
8
i—
Q)
CL
100
-100
-200
Temperature=25°C, Humidity=45%
'' oTa
o
oj?
s
O
O
£
A
Carpet
Paper
Steel
Wood
Plastic Rubber
Material
O Crude, 7 Days O Pure, 7 Days A Crude, 14 Days LI Pure, 14 Days
Figure B-1d. Geometric Mean Percent Reduction, 95% Confidence Intervals, and
Observed Percent Reduction Plotted by Material and Ricin Type for Days 7
and 14, 25 °C and 45% Humidity.
B-46
-------
Temperature=25°C. Humidity=75%
100
0
c
o
-100
b
"O
-200
CD
a:
C
-300
8
Cl>
-400
CL
-500
-600
C, Humidity=45%
c
o
o
is
T3
Q)
a:
-4—•
c
8
a)
Q_
100
0
-100
-200
-300
-400
-500
-600
-700
-800
-900
-1000
Figure B-1f.
O
-r
Carpet
Paper
Steel
Wood
C Crude, 7 Days
Plastic Rubber
Material
O Pure, 7 Days A Crude, 14 Days LJ Pure, 14 Days
Geometric Mean Percent Reduction, 95% Confidence Intervals, and
Observed Percent Reduction Plotted by Material and Ricin Type for Days 7
and 14, 30 °C and 45% Humidity.
B-47
-------
c
o
o
100
0
-100
-200
~o
CD
a: -3oo
c
8
i—
0
CL
-400
-500
-600
-700
Temperature=30"C. Humidity=75%
a
^ ^5 LJ c
- §-
Carpet Paper Plastic Rubber
Material
Steel
Wood
O Crude, 7 Days 0 Pure, 7 Days A Crude, 14 Days Pure, 14 Days
Figure B-1g. Geometric Mean Percent Reduction, 95% Confidence Intervals, and
Observed Percent Reduction Plotted by Material and Ricin Type for Days 7
and 14, 30 °C and 75% Humidity.
100
c
o
-~—«
0
"D "100
Q)
o:
1 -200
o
CD
CL
-300
-400
Temperature=40'>C, Humidity=20%
0^
-
O
IT i
&- !
1 8
0= Q 0= P
t-
o
O
¥
*4
8 jf"
o
48 72 96 120 144
Hours Hours Hours Hours Hours
7
Days
Time
10
Days
11 12
Days Days
13
Days
14
Days
O Crude O Pure
Figure B-1h. Geometric Mean Percent Reduction, 95% Confidence Intervals, and
Observed Percent Reduction Plotted by Ricin Type and Time for Steel, 40
°C and 20% Humidity,
B-48
-------
UU :
90 .
80-
70 :
60-
50 ;
40-
30:
20 i
10
0
-10-
-1i,
Temperature=50"C. Humidity=20%
O
.. "
o
O"
•f
«•
o
o
O 0"
o
o-
o
8"
o
6
o
0-
o
o
o"
o
o
0- G=0-
6
Hours
24
Hours
30
Hours
48
Hou rs
72
Hours
Time
96
Hours
120
Hours
144
Hours
7
Days
O Crude O Pure
Geometric Mean Percent Reduction, 95% Confidence Intervals, and
Observed Percent Reduction Plotted by Ricin Type and Time for Steel, 50
°C and 20% Humidity.
B-49
-------
Temperature=20°C, 7 Days
100
0
.I "10°
"o
=3 -200
TJ
CD
a: -3oo
c
a; -4oo
0
q_ -500
-600
-700
B
©-
f
A-
A
0=
Hf 8-
o
s 5.
0
Q
O
A ~
~
~
Carpet Paper Plastic Rubber
Material
Steel
Wood
O Crude, 45% O Crude, 75% A Pure, 45% U Pure, 75%
Figure B-2a.
Geometric Mean Percent Reduction, 95% Confidence Intervals, and
Observed Percent Reduction Plotted by Material and Ricin Type for Day 7
and 20 °C, 45% and 75% Humidity.
Temperature=25'JC 7 Days
100-
0
c
o
o
Z5
T3
CD
q; -100
c
8
0
~l
-200
-300
nNE if
T
A
o
5
at 0-
&
0
STi a
o
Carpet Paper Plastic Rubber
Material
Steel
Wood
O Crude, 45% O Crude, 75% A Pure, 45% ~ Pure, 75%
Figure B-2b. Geometric Mean Percent Reduction, 95% Confidence Intervals, and
Observed Percent Reduction Plotted by Material and Ricin Type for Day 7
and 25 °C, 45% and 75% Humidity.
B-50
-------
Temperature=30t'C, 7 Days
C
o
o
3
T3
CD
Dd
-4—'
C
8
i—
0
CL
100
0
-100
-200
-300
-400
-500
-600
-700
-800
-900
-1000
Qe Z5r
°#f
§ 0= 'Vtj- ^ ^~=
o
8
o
Carpet Paper Plastic Rubber
Material
Steel
Wood
O Crude, 45% 0 Crude, 75% A Pure, 45% U Pure, 75%
Figure B-2c. Geometric Mean Percent Reduction, 95% Confidence Intervals, and
Observed Percent Reduction Plotted by Material and Ricin Type for Day 7
and 30 °C, 45% and 75% Humidity.
Temperature=20°C, 14 Days
C
o
o
Z5
~o
CD
Dd
8
¦—
CD
~l
100-
0
-100
-200
-300
8J-J-
o
¦- i.
4P-
O-
o
o
"Hi
O
. A
it
O
O
o-
o
o
J
Carpet Paper Plastic Rubber
Material
Steel
Wood
O Crude, 45% O Crude, 75% A Pure, 45% ~ Pure, 75%
Figure B-2d. Geometric Mean Percent Reduction, 95% Confidence Intervals, and
Observed Percent Reduction Plotted by Material and Ricin Type for Day 14
and 20 °C, 45% and 75% Humidity.
B-51
-------
Temperature=25°C, 14 Days
100
0
-100
-200
-300
C
o
o
T3
CD
Dd
-»—»
C
8
0 -400
CL
-500
-600
^=3= QbC*5 ~
J
a- Qe
0- £p^--
%
e.
Carpet Paper Plastic Rubber
Material
Steel
Wood
O Crude, 45% O Crude, 75% A Pure, 45% U Pure, 75%
Figure B-2e. Geometric Mean Percent Reduction, 95% Confidence Intervals, and
Observed Percent Reduction Plotted by Material and Ricin Type for Day 14
and 25 °C, 45% and 75% Humidity.
Temperature=30°C, 14 Days
C
o
o
Z5
~o
CD
Dd
-+—»
c
8
i—
0
~L
100-
0
-100
-200
-300
-400
-500
O
~
~
Carpet Paper Plastic Rubber
Material
Steel
Wood
O Crude, 45% O Crude, 75% A Pure, 45% ~ Pure, 75%
Figure B-2f. Geometric Mean Percent Reduction, 95% Confidence Intervals, and
Observed Percent Reduction Plotted by Material and Ricin Type for Day 14
and 30 °C, 45% and 75% Humidity.
B-52
-------
vvEPA
United States
Environmental Protection
Agency
PRESORTED STANDARD
POSTAGE & FEES PAID
EPA
PERMIT NO. G-35
Office of Research and Development (8101R)
Washington, DC 20460
Official Business
Penalty for Private Use
$300
------- |